Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling

ABSTRACT

Methods for preparing oligonucleotide analogs which have improved nuclease resistance and improved cellular uptake are provided. In preferred embodiments, the methods involve reductive coupling of 3&#39;- and 4&#39;-substituted or 4&#39;- and 3&#39;-substituted nucleosidic synthons.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US92/04294, filed May21, 1992, (now U.S. Ser. No. 08/150,079, filed Apr. 7, 1994) and of U.S.Ser. No. 903,160, filed Jun. 24, 1992, which are continuations-in-partof U.S. Ser. No. 703,619 filed May 21, 1991, which is acontinuation-in-part of Ser. No. 566,836, filed Aug. 13, 1990, U.S. Pat.No. 5,223,618, issued Jun. 29, 1993, and U.S. Ser. No. 558,663 filedJul. 27, 1990, U.S. Pat. No. 5,138,045, issued Aug. 11, 1992. Thisapplication also is related to the subject matter disclosed and claimedin the following patent application filed by the present inventors: U.S.Ser. No. 039,979, filed Mar. 30, 1993; U.S. Ser. No. 040,526, filed Mar.31, 1993; and U.S. Ser. No. 040,993, filed Mar. 31, 1993.

Each of these patent applications are assigned to the assignee of thisapplication and are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant oligonucleotide analogs which are useful fordiagnostics and as research reagents. Oligonucleotide analogs areprovided having modified linkages replacing the phosphorodiester bondsthat normally serve as inter-sugar linkages in wild type nucleic acids.Such analogs are resistant to nuclease degradation and are capable ofmodulating the activity of DNA and RNA. Methods for synthesizing theseoligonucleotide analogs and for modulating the production of proteinsare also provided, as are intermediate compositions and syntheticmethods.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are effected by proteins. Proteins, either actingdirectly or through their enzymatic functions, contribute in majorproportion to many diseases in animals and man.

Classical therapeutics generally has focused upon interactions withproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made tomoderate the production of proteins by interactions with the molecules(i.e., intracellular RNA) that direct their synthesis. Theseinteractions have involved hybridization of complementary "antisense"oligonucleotides or certain analogs thereof to RNA. Hybridization is thesequence-specific hydrogen bonding of oligonucleotides oroligonucleotide analogs to RNA or to single stranded DNA. By interferingwith the production of proteins, it has been hoped to effect therapeuticresults with maximum effect and minimal side effects.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogs, like other therapeutics, depends on a number offactors that influence the effective concentration of these agents atspecific intracellular targets. One important factor foroligonucleotides is the stability of the species in the presence ofnucleases. It is unlikely that unmodified oligonucleotides will beuseful therapeutic agents because they are rapidly degraded bynucleases. Modification of oligonucleotides to render them resistant tonucleases therefore is greatly desired.

Modification of oligonucleotides to enhance nuclease resistancegenerally has taken place on the phosphorus atom of the sugar-phosphatebackbone. Phosphorothioates, methyl phosphonates, phosphoramidates andphosphorotriesters have been reported to confer various levels ofnuclease resistance. Phosphate-modified oligonucleotides, however,generally have suffered from inferior hybridization properties. See,e.g., Cohen, J. S., ed Oligonucleotides: Antisense Inhibitors of GeneExpression, (CRC Press, Inc., Boca Raton, Fla., 1989).

Another key factor is the ability of antisense compounds to traverse theplasma membrane of specific cells involved in the disease process.Cellular membranes consist of lipid-protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and are inherentlyimpermeable to most natural metabolites and therapeutic agents. See,e.g., Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological andantiviral effects of natural and modified oligonucleotides in culturedmammalian cells have been well documented. It appears that these agentscan penetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has beenstudied using natural oligonucleotides and certain nuclease resistantanalogs, such as alkyl triesters and methyl phosphonates. See, e.g.,Miller, et al., Biochemistry 1977, 16, 1988; Marcus-Sekura, et al.,Nucleic Acids Research 1987, 15, 5749; and Loke, et al., Top. Microbiol.Immunol. 1988, 141, 282.

Often, modified oligonucleotides and oligonucleotide analogs areinternalized less readily than their natural counterpart. As a result,the activity of many previously available antisense oligonucleotides hasnot been sufficient for practical therapeutic, research or diagnosticpurposes. Two other serious deficiencies of prior art compounds designedfor antisense therapeutics are inferior hybridization to intracellularRNA and the lack of a defined chemical or enzyme-mediated event toterminate essential RNA functions.

Modifications to enhance the effectiveness of the antisenseoligonucleotides and overcome these problems have taken many forms.These modifications include base ring modifications, sugar moietymodifications and sugar-phosphate backbone modifications. Priorsugar-phosphate backbone modifications, particularly on the phosphorusatom, have effected various levels of resistance to nucleases. However,while the ability of an antisense oligonucleotide to bind to specificDNA or RNA with fidelity is fundamental to antisense methodology,modified phosphorus oligonucleotides have generally suffered frominferior hybridization properties.

Replacement of the phosphorus atom has been an alternative approach inattempting to avoid the problems associated with modification on thepro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters1990, 31, 2385 disclosed the replacement of the phosphorus atom with amethylene group. However, this replacement yielded unstable compoundswith nonuniform insertion of formacetal linkages throughout theirbackbones. Cormier, et al., Nucleic Acids Research 1988, 16, 4583,disclosed replacement of phosphorus with a diisopropylsilyl moiety toyield homopolymers having poor solubility and hybridization properties.Stirchak, et al., Journal of Organic Chemistry 1987, 52, 4202 disclosedreplacement of phosphorus linkages by short homopolymers containingcarbamate or morpholino linkages to yield compounds having poorsolubility and hybridization properties. Mazur, et al., Tetrahedron1984, 40, 3949, disclosed replacement of a phosphorus linkage with aphosphonic linkage yielded only a homotrimer molecule. Goodchild,Bioconjugate Chemistry 1990, 1, 165, disclosed ester linkages that areenzymatically degraded by esterases and, therefore, are not suitable forantisense applications.

The limitations of available methods for modification of the phosphorusbackbone have led to a continuing and long felt need for othermodifications which provide resistance to nucleases and satisfactoryhybridization properties for antisense oligonucleotide diagnostics andtherapeutics.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligonucleotide analogs fordiagnostic and research use.

It is a further object of the invention to provide oligonucleotideanalogs having enhanced cellular uptake.

Another object of the invention is to provide oligonucleotide analogshaving greater efficacy than unmodified oligonucleotides.

It is yet another object of the invention to provide methods forsynthesis and use of oligonucleotide analogs.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification and theappended claims.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that mimic and/ormodulate the activity of wild-type nucleic acids. In general, thecompounds contain a selected nucleoside sequence which is specificallyhybridizable with a targeted nucleoside sequence of single stranded ordouble stranded DNA or RNA. At least a portion of the compounds of theinvention has structure I: ##STR1## wherein L₁ --L₂ --L₃ --L₄ is CH₂--R_(A) --NR₁ --CH₂, CH₂ --NR₁ --R_(A) --CH₂, R_(A) --NR₁ --CH₂ --CH₂ orNR₁ --R_(A) --CH₂ --CH₂ ;

R_(A) is O or NR₂ ;

R₁ and R₂ are the same or different and are H; alkyl or substitutedalkyl having 1 to about 10 carbon atoms; alkenyl or substituted alkenylhaving 2 to about 10 carbon atoms; alkynyl or substituted alkynyl having2 to about 10 carbon atoms; alkaryl, substituted alkaryl, aralkyl, orsubstituted aralkyl having 7 to about 14 carbon atoms; alicyclic;heterocyclic; a reporter molecule; an RNA cleaving group; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of anoligonucleotide;

B_(x) is a nucleosidic base;

n is an integer greater than 0;

Q is O, S, CH₂, CHF or CF₂ ;

X is H; OH; alkyl or substituted alkyl having 1 to about 10 carbonatoms; alkaryl, substituted alkaryl, aralkyl, or substituted aralkylhaving 7 to about 14 carbon atoms; F; Cl; Br; CN; CF₃ ; OCF₃ ; OCN;O-alkyl; S-alkyl; N-alkyl; O-alkenyl; S-alkenyl; N-alkenyl; SOCH₃ ; SO₂CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino or substituted silyl; an RNA cleavinggroup; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an aligonucleotide.

The compounds of the invention are prepared by coupling preselected3'-functionalized and 4'-functionalized nucleosides and/oroligonucleotides under conditions effective to form the above-noted L₁--L₂ --L₃ --L₄ linkages. In certain embodiments, a 3'-formyl nucleosideor oligonucleotide synthon is reacted with a 5'-hydroxylamino or5'-hydrazino nucleoside or oligonucleotide synthon. In otherembodiments, a 5'-formyl synthon is reacted with a3'-methylhydroxylamino or 3'-methylhydrazino synthon. In still furtherembodiments, linkages having structure CH═N--R_(A) --CH₂, CH₂--CH═N--R_(A), CH₂ --R_(A) --N═CH, or R_(A) --N═CH--CH₂ where R_(A) is Oor NR₁ are formed by coupling synthons having structures II and III:##STR2## wherein: Z₁ and Y₂ are selected such that

(i) Z₁ is C(O)H and Y₂ is CH₂ R_(A) NH₂ ; or

Z₁ is CH₂ R_(A) NH₂ and Y₂ is C(O)H;

(iii) Z₁ is CH₂ C(O)H and Y₂ is R_(A) NH₂ ; or

(iv) Z₁ is R_(A) NH₂ and Y₂ is H(O)CCH₂ ;

Y₁ is aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate,methylalkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof;

Z₂ is hydroxyl, aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxy-

Z₂ is hydroxyl, aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylamino-benzenethio, methylphosphonate,methylalkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof;

B_(X1) and B_(X2) are, independently, nucleosidic bases; Q₁ and Q₂ are,independently, O, S, CH₂, CHF or CF₂ ; and

X₁ and X₂ are, independently, H, OH, alkyl, substituted alkyl, alkarylor aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide.

In preferred embodiments, the resulting compounds are reduced to producethe linkages CH₂ --NH--R_(A) --CH₂, CH₂ --R_(A) --NH--CH₂, or R_(A)--NH--CH₂ --CH₂, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic, synthetic scheme showing the synthesis of anoligonucleoside linkage containing adjacent nitrogen atoms.

FIG. 2 is a schematic, synthetic scheme showing the synthesis of anoligonucleoside linkage containing adjacent oxygen and nitrogen atoms.

DETAILED DESCRIPTION OF THE INVENTION

The term "nucleoside" as used in connection with this invention refersto a unit made up of a heterocyclic base and its sugar. The term"nucleotide" refers to a nucleoside having a phosphate group on its 3'or 5' sugar hydroxyl group. Thus nucleosides, unlike nucleotides, haveno phosphate group. "Oligonucleotide" refers to a plurality of joinednucleotide units formed in a specific sequence from naturally occurringbases and pentofuranosyl groups joined through a sugar group by nativephosphodiester bonds. This term refers to both naturally occurring andsynthetic species formed from naturally occurring subunits.

The compounds of the invention generally can be viewed as"oligonucleotide analogs", that is, compounds which function likeoligonucleotides but which have non-naturally occurring portions.Oligonucleotide analogs can have altered sugar moieties, altered basemoieties or altered inter-sugar linkages. For the purposes of thisinvention, an oligonucleotide analog having non-phosphodiester bonds,i.e., an altered inter-sugar linkage, is considered to be an"oligonucleoside." The term "oligonucleoside" thus refers to a pluralityof nucleoside units joined by linking groups other than nativephosphodiester linking groups. The term "oligomers" is intended toencompass oligonucleotides, oligonucleotide analogs or oligonucleosides.Thus, in speaking of "oligomers" reference is made to a series ofnucleosides or nucleoside analogs that are joined via either naturalphosphodiester bonds or other linkages, including the four atom linkersof this invention. Although the linkage generally is from the 3' carbonof one nucleoside to the 5' carbon of a second nucleoside, the term"oligomer" can also include other linkages such as 2'-5' linkages.

Oligonucleotide analogs also can include other modifications consistentwith the spirit of this invention, particularly modifications thatincrease nuclease resistance. For example, when the sugar portion of anucleoside or nucleotide is replaced by a carbocyclic moiety, it is nolonger a sugar. Moreover, when other substitutions, such a substitutionfor the inter-sugar phosphorodiester linkage are made, the resultingmaterial is no longer a true nucleic acid species. All such compoundsare considered to be analogs. Throughout this specification, referenceto the sugar position of a nucleic acid species shall be understood torefer to either a true sugar or to a species taking the structural placeof the sugar or wild type nucleic acids. Moreover, reference tointer-sugar linkages shall be taken to include moieties serving to jointhe sugar or sugar analog portions in the fashion of wild type nucleicacids.

This invention concerns modified oligonucleotides, i.e., oligonucleotideanalogs or oligonucleosides, the methods for effecting themodifications. These modified oligonucleotides and oligonucleotideanalogs exhibit increased stability relative to their naturallyoccurring counterparts. Extracellular and intracellular nucleasesgenerally do not recognize and therefore do not bind to thebackbone-modified compounds of the invention. In addition, the neutralor positively charged backbones of the present invention can be takeninto cells by simple passive transport rather than by complicatedprotein-mediated processes. Another advantage of the invention is thatthe lack of a negatively charged backbone facilitates sequence specificbinding of the oligonucleotide analogs or oligonucleosides to targetedRNA, which has a negatively charged backbone and will repel similarlycharged oligonucleotides. Still another advantage of the presentinvention is it presents sites for attaching functional groups thatinitiate cleavage of targeted RNA.

The modified internucleoside linkages of this invention preferablyreplace naturally-occurring phosphodiester-5'-methylene linkages withfour atom linking groups to confer nuclease resistance and enhancedcellular uptake to the resulting compound. Preferred linkages havestructure CH₂ --R_(A) --NR₁ --CH₂, CH₂ --NR₁ --R_(A) --CH₂, R_(A) --NR₁--CH₂ --CH₂, or NR₁ --R_(A) --CH₂ --CH₂ where R_(A) is O or NR₂.

Generally, these linkages are prepared by functionalizing the sugarmoieties of two nucleosides which ultimately are to be adjacent to oneanother in the selected sequence. In a 4' to 3' sense, an "upstream"synthon such as structure II is modified at its terminal 3' site, whilea "downstream" synthon such as structure III is modified at its terminal4' site. More specifically, the invention provides efficient synthesesof oligonucleosides via intermolecular reductive coupling.

B_(X1) and B_(X2) can be nucleosidic bases selected from adenine,guanine, uracil, thymine, cytosine, 2-amonoadenosine or5-methylcytosine, although other non-naturally occurring species can beemployed to provide stable duplex or triplex formation with, forexample, DNA. Representative bases are disclosed in U.S. Pat. No.3,687,808 (Merigan, et al.), which is incorporated herein by reference.

Q₁ and Q₂ can be S, CH₂, CHF CF₂ or, preferably, O. See, e.g., Secrist,et al., Abstract 21, Synthesis and Biological Activity of4'-Thionucleosides, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sept. 16-20, 1992.

X₁ and X₂ are, independently, H, OH, alkyl, substituted alkyl, alkarylor aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide. It isintended that the term "alkyl" denote branched and straight chainhydrocarbyl residues, including alkyl groups having one or more ³ Hand/or ¹⁴ C atoms. It is preferred that X is H or OH, or, alternativelyF, O-alkyl or O-alkenyl, especially where Q is O. Preferred alkyl andalkenyl groups have from 1 to about 10 carbon atoms.

Y₁ and Z₂ can be aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl-substituted imidazolidino,aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate,methyl-alkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide,an oligonucleoside, or a hydroxyl-protected or amine-protectedderivative thereof. In addition, Z₂ can be hydroxyl. Preferably, Y₁ is aprotected hydroxymethyl group or a nucleoside or oligonucleosideattached by, for example, a phosphodiester-5'-methylene linkage or someother four atom linking group, and Z₂ is a protected hydroxyl group or anucleoside or oligonucleoside attached by, for example, aphosphodiester-3'-hydroxyl linkage or some other four atom linkinggroup.

It is preferred that the oligonucleotide analogs of the inventioncomprise from about 5 to about 50 subunits having the given structure(i.e., n=5-50). While each subunit of the oligonucleotide analogs canhave repetitive structure I, such need not be the case. For example, thesubunits can have alternating or more random structures.

The invention is also directed to methods for the preparation ofoligonucleosides with modified inter-sugar linkages. These modificationsmay be effected using solid supports which may be manually manipulatedor used in conjunction with a DNA synthesizer using methodology commonlyknown to those skilled in DNA synthesizer arts. Generally, the procedureinvolves functionalizing the sugar moieties of two nucleosides whichwill be adjacent to one another in the selected sequence. In a 5' to 3'sense, an "upstream" synthon such as structure II is modified at itsterminal 3' site, while a "downstream" synthon such as structure III ismodified at its terminal 5' site.

More specifically, certain linkages can be formed by selecting a3'-C-formyl derivatized compound as the upstream synthon and a5'-aminohydroxy derivatized compound as the downstream synthon. Couplingthen is effected to provide, for example, a dinucleoside having an oximelinkage. In this instance, the oxime is present as E/Z isomers, whichare separated by HPLC. The oxime nitrogen atom is adjacent to a carbonatom on the 3' end of the upstream nucleoside. Dinucleosides having theoxime nitrogen adjacent to a carbon atom on the 5' or downstreamnucleoside are synthesized utilizing a 5'-C-formyl derivatized compoundas the upstream synthon and a 3'-deoxy-3'-aminohydroxymethyl derivatizedcompound as the downstream synthon, again providing E/Z isomers. In bothinstances the oxime linked compound can be incorporated directly into anoligomer and/or can be reduced to a corresponding hydroxyamino linkedspecies. Reduction of oxime linked dinucleosides either as thedinucleoside or as a dinucleoside moiety in an oligomer with sodiumcyanoborohydride yields the corresponding hydroxylamino linkedcompounds. Hydroxylamino linked compounds can be alkylated at the aminomoiety of the hydroxylamino linkage to yield a correspondingN-alkylamino linkage.

3'-C-formyl derivatized nucleosides can be formed via several syntheticpathways. The presently preferred methods utilizes a radicalcarbonylation of the corresponding 3'-deoxy-3'-iodo nucleoside. The iodocompound is treated with CO, 2,2'-azobisisobutrylonitrile (AIBN), andtris(trimethylsilyl)silane (TTMS). Alternately, 3'-C-formyl derivatizedcompounds can be synthesized from either a 3'-deoxy-3'-cyano sugar ornucleoside. Both 5'-C-formyl (also identified as 5'-aldehydo) and3'-C-formyl group an be blocked in a facile manner utilizingo-methylaminobenzenthiol as a blocking group. The 5'-and 3'-C-formylgroups can be deblocked with silver nitrate oxidation.

An alternate method of 3'-C-formyl nucleoside synthesis employs1-O-methyl-3'-deoxy-3'-O-methylaminobenzenethiol-5'-O-trityl-β-D-erythro-pento furanoside, which serves as aprecursor for any 3'-deoxy-3'-C-formyl nucleoside. The1-O-methyl-3'-deoxy-3'-O-methyl aminobenzenethiol-5'-O-trityl-β-D-erythro-pentofuranoside is reacted with anappropriate base utilizing standard glycosylation conditions and thendeblocked to yield the nucleoside. In yet another method, a3'-deoxy-3'-cyano nucleoside is prepared from either the corresponding3'-deoxy-3'-iodo nucleoside or by glycosylation with1-O-methyl-3'-deoxy-3'-O-cyano-5'-O-trityl-β-D-erythro-pentofuranoside.

Resulting dinucleosides from any of the above described methods, linkedby hydrazines, hydroxyl amines and other linking groups, can beprotected by a dimethoxytrityl group at the 5'-hydroxyl and activatedfor coupling at the 3'-hydroxyl with cyanoethyldiisopropyl-phosphitemoieties. These dimers can be inserted into any desired sequence bystandard, solid phase, automated DNA synthesis utilizing phosphoramiditecoupling chemistries. The protected dinucleosides are linked with theunits of a specified DNA sequence utilizing normal phosphodiester bonds.The resulting oligonucleotide analog or oligomer has a "mixed" backbonecontaining both phosphodiester linkages and four atoms linkages of theinventions. In tis manner, a sequence-specific 15-mer oligonucleotidecan be synthesized to have seven hydroxylamine, hydrazine or other typelinked dinucleosides attached via alternating phosphodiester linkages.Such a structure will provide increased solubility in water compared tofully modified oligomers, which may contain linkages of the invention.

Oligonucleosides containing a uniform backbone linkage can besynthesized by use of CPG-solid support and standard nucleic acidsynthesizing machines such as Applied Biosystems Inc. 380B and 394 andMilligen/Biosearch 7500 and 8800s. The initial nucleoside (number 1 atthe 3'-terminus) is attached to a solid support such as controlled poreglass. In sequence specific order, each new nucleoside is attachedeither by manual manipulation or by the automated synthesizer system. Inthe case of a methylenehydrazine linkage, the repeating nucleoside unitcan be of two general types--a nucleoside with a 5'-protected aldehydicfunction and a 3'-deoxy-3'-C-hydrazinomethyl group, or a nucleosidebearing a 5'-deoxy-5'-hydrazino group protected by an acid labile groupand a 2'-deoxy-3'-C-formyl group. In each case, the conditions which arerepeated for each cycle to add the subsequent sequence required baseinclude: acid washing to remove the 5'-aldehydo protecting group;addition of the next nucleoside with a 3'-methylenehydrazino group toform the respective hydrazone connection; and reduction with any of avariety of agents to afford the desired methylene-hydrazine linkedCPG-bound oligonucleosides. One such useful reducing agent is sodiumcyanoborohydride.

A preferred method is depicted in FIG. 1. This method employs a solidsupport to which has been found a downstream synthon having a protected5' site. Preferably, the 5' site of said synthon is protected with DMT.Thereafter, the 5' site of the synthon is liberated with mild acid,washed, and oxidized to produce an intermediate product. In onepreferred method, the aldehyde derivative reacts withN,N-diphenylethylene diamine to produce an intermediary product, a5'-diphenylimidazolidino protected synthon. In a more preferred methodthe 5'-diphenylimidazolidino protected synthon is directly loaded on thesupport. With either method, the intermediary product can besubsequently deblocked to provide a synthon with a nucleophilic 5'position.

An upstream synthon having a protected 5'-aldehyde group, such as a5'-diphenylimidazolidino protected 3'-deoxy-3'-C-hydrazine base, iscoupled wit the bound downstream synthon by, for example, the additionof sodium cyanoborohydride. Following a wash step, a dinucleoside linkedthrough a hydrazino moiety is formed. Thereafter, the cycle can berepeated by the addition of an upstream synthon, followed by acid/ basedeprotection to create a polymeric synthon of a desired sequencecontaining modified inter-sugar linkages. In some preferred embodimentsof this invention, the upstream synthon is a 5'-DMT protected3'-C-hydrazine base.

One preferred process employs a diphenylethyldiamine adduct(1,3-disubstituted imidazolidino) to protect the electrophilic center ofthe downstream synthon during attachment to the solid support. Moffatt,et al., J. Am. Chem. Soc. 1968, 90, 5337. The downstream synthon can beattached to a solid support such as a controlled pore glass support orother suitable supports known to those skilled in the art. Attachmentcan be effected via standard procedures. Gait, M. J., ed.,Oligonucleotide Synthesis, A Practical Approach (IRL Press 1984).Alternatively, the protected bound nucleoside can be oxidized bystandard oxidizing procedures. Bound downstream synthons preferably arereacted with hydrazine to produce a Schiff's base, which can be reduced.Hydroxylamine is also a preferred reactant in this method.

A further method of synthesizing uniform backbone linkedoligonucleosides is depicted in FIG. 2. This method also employs a solidsupport to which has been bound a downstream synthon with a protected 5'site. The 5' site preferably is protected with a phthalimido group. The5' site of the downstream synthon is liberated with methylhydrazine inDCM and washed with DCM:methanol. The aminohydroxyl group at the 5'position of the upstream synthon also is protected with a phthalimidogroup to yield a 5'-phthalimido protected 3'-deoxy-3'-C-formylnucleoside, which is reacted with the downstream synthon. Deprotectionat the 5' position and washing liberates the next 5'-aminohydroxyreaction site. The cycle is repeated with the further addition ofupstream synthon until the desired sequence is constructed. Eachnucleoside of this sequence is connected with oxime linkages. Theterminal nucleoside of the desired oligonucleoside is added to thesequence as a 5'-OTBDMSi blocked 3'-deoxy-3'-C-formyl nucleoside. Theoxime linked oligonucleoside can be removed from the support. If aaminohydroxyl linked oligonucleoside is desired, the oxime linkages arereduced with sodium cyanoborohydride. Alternately reduction can beaccomplished while the oxime linked oligonucleoside is still connectedto the support.

The reactions of Examples 25-27 represent improved syntheses of3'--O--NH₂ nucleosides. In forming --O--NH₂ moieties on sugars, it istheoretically possible to displace a leaving group, as for instance atosyl group, with hydroxylamine. However, Files et al., J. Am. Chem.Soc. 1992, 14, 1493 have shown that such a displacement leads to apreponderance of --NHOH moieties and not to the desired --O--NH₂moieties. Further, the reaction sequence of Examples 25-27 represents animproved synthesis compared to that illustrated in European PatentApplication 0 381 335. The synthetic pathway of that patent applicationrequires the use of a xylo nucleoside as the staring material. Xylonucleosides are less readily obtainable than the ribonucleoside utilizedin Examples 25-27.

The methods of the invention can be modified for use with eithersolution-phase or solid-phases techniques. For example, the compounds ofthe invention can be synthesized using controlled pore glass (CPG)supports and standard nucleic acid synthesizing machines such as AppliedBiosystems Inc. 380B and 394 and Milligen/Biosearch 7500 and 8700s. Eachnew nucleoside is attached either by manual manipulation or by automatedtechniques.

A wide variety of protecting groups can be employed in the methods ofthe invention. See, e.g., Beaucage, et al., Tetrahedron 1992, 12, 2223.In general, protecting groups render chemical functionality inert tospecific reaction conditions, and can be appended to and removed fromsuch functionality in a molecule without substantially damaging theremainder of the molecule. Representative hydroxyl protecting groupsinclude t-butyldimethylsilyl (TBDMSi), t-butyldiphenylsilyl (TBDPSi),dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and other hydroxylprotecting groups as outlined in the above-noted Beaucage reference.

Scheme I illustrates the conversion of a 4'-aldehydo nucleoside to a5'-aldehydo nucleoside. This reaction is exemplified in Example 31.Scheme II illustrates the generation of a 5'-aldehydo methyl sugar. Thises exemplified in Example 29. Scheme III illustrates the formation of an5'-iodo nucleoside. Similar methodology is used to generate an activeiodo group on a terminal hydroxyl of a dimeric unit in Scheme IX. InScheme III, the iodo nucleoside is further derivatized to a 6'-aldehydonucleoside via an allyl substituted nucleoside. This is exemplified inExamples 46 and 47.

Scheme IV illustrates a free radical reaction of a --O--methyleneaminonucleoside of Scheme 1 to a 5'-amino 5'-homo nucleoside. This isexemplified in Example 45. Scheme V illustrates use of a Mitsunobureaction on a 5'-homo nucleoside to synthesize an oxyaminehomonucleoside, i.e. a 6'--O--NH₂ 5'-homo nucleoside. This isexemplified in Examples 49, 50, and 51. Scheme VI illustratesN-alkylation of the amino moiety of a 6'-amino-5'-deoxy-5'-homonucleoside. This is exemplified in Examples 56, 57, and 58. SuchN-alkylation is desirable where the amino moiety subsequently will bereacted with a thiol moiety. The N-alkylated product of such a reactionexhibits greater stability to acid than does the non-alkylated S--Nbond. This is particularly useful in solid support synthesis whereinacid removal of trityl groups is commonly practiced. However, for othersynthesis, such as solution synthesis, this may not be a concern.

Schemes VII to XV illustrate the use of nucleosides for the assembly ofdimeric, trimeric and other, higher order oligonucleosides. In SchemeVII, nucleosides 3 and 31 are joined via an acid catalyzed couplingreaction to form an --O--nitrilomethylidyne linkage between therespective two nucleosides. This is exemplified in Example 32. Dimericoligonucleoside 32 can be reduced to an iminomethylene linkage that, inturn, can be alkylated to a (methylimino)methylene linkage, asexemplified in Example 33.

Scheme VIII illustrates the coupling of nucleoside 3 to nucleoside 5.This scheme is analogous to Scheme VII with the exception that in SchemeVII a three atom linkage is created whereas in Scheme VII a four atomlinkage is created. Nucleosides 3 and 5 are joined in Step 1 to form an--O--nitrilo linkage that is reduced in Step 2 to an --O--imino linkage.Alkylation occurs in Step 3 to a --O--methylimino linkage, with finaldeblocking in Step 4. These steps are exemplified in Example 28. Thealkylation reaction in Step 3 is accompanied by deblocking thet-butyldimethylsilyl protecting group at the 5' terminus of the dimer.Advantageous use of this deblocking reaction also is utilized in otherSchemes. Deblocking of the t-butyldiphenylsilyl group used to protectthe 3' terminus of the dimer is effected using tetra-n-butylammoniumfluoride.

The alkylation step can be used to introduce other, useful, functionalmolecules on the macromolecule. Such useful functional molecules includebut are not limited to reporter molecules, RNA cleaving groups, groupsfor improving the pharmacokinetic properties of an oligonucleotide, andgroups for improving the pharmacodynamic properties of anoligonucleotide. Such molecules can be attached to or conjugated to themacromolecule via attachment to the nitrogen atom in the backbonelinkage. Alternatively, such molecules can be attached to pendent groupsextending from the 2' position of the sugar moiety of one or more of thenucleosides of the marcromolecules. Examples of such other usefulfunctional groups are provided by U.S. patent application Ser. No.782,374, filed Oct. 24, 1991, entitled Derivatized OligonucleotidesHaving Improved Uptake & Other Properties, assigned to the same assigneeas this application, herein incorporated by reference, and any other ofthe above-referenced patent applications.

Scheme IX illustrates a synthetic scheme utilized to prepare dimers,trimers, and other, higher order oligonucleosides having homogenouslinkages between nucleosides. In this scheme, nucleosides 10 and 12 arelinked to form an iminomethylene linkage. Advantageous use of thealkylating-5' terminus deblocking step of Scheme VII is effected toremove the blocking group at the 5' terminus of the dimericoligonucleoside 14. Using the iodination reaction of Scheme III, thedimer is then converted to a 5' terminus iodo intermediate. A further3'--O--methyleneamino nucleosidic unit 10 then can be added to the dimerto form a trimer, followed by deblocking and alkylation. This reactionsequence can be repeated any number of times to form a higher orderoligonucleoside. The oligonucleoside is deblocked at the 3' terminus.

Scheme X illustrates the use of an 1--O--alkyl sugar that is firstlinked to a nucleoside. Reduction followed by alkylation and deblockingyields an --O--(methylimino)methylene linkage joining the 1--O--alkylsugar and the nucleoside, as exemplified by Example 34. This structureis then blocked at the 5' terminus, as exemplified by Example 35. Thefully blocked, linked sugar-nucleoside structure is then subjected toglycosylation to add a hetrocyclic base to the sugar moiety and thusform a dimeric nucleoside structure, as in Example 36. Afterglycosylation, removal of the 5' terminus blocking group andchromatographic separation of α and β anomers, as exemplified by Example37, yields a dimer. This dimer can be further elongated as per theprocedure of Scheme IX. Examples 39 and 40 exemplify the addition of anadenine, cytosine and guanine base to a thymidine-methyl sugar dimer toform T-A, T-C and T-G dimers in addition to the T-T dimer of Scheme IX.Examples 41, 42, and 43 exemplify the formation of A-T, A-A, A-C, A-G,C-T, C-A, C-C, C-G, G-T, G-A, G-C and G-G dimers. Each may be furtherelongated as per the procedures of Scheme IX.

Scheme XI illustrates the formation of an imino-oxymethylene linkage.Example 48 describes the preparation of the 5'--O--trityl protected xylostarting nucleoside and Example 52 describes the reaction of compound 50with compound 54 to form a dimeric unit Continuing within Scheme XI, toprepare dimeric units that can be used as solid support building blocks(Example 53), the backbone nitrogen atom is alkylated, followed bysimultaneous removal of both the 5'--O--trityl and the3'--O--(t-butyldiphenylsilyl) protecting groups with trifluoraceticacid. The 5'-terminus hydroxyl group is blocked with dimethoxytriryl(Example 54), followed by forming an active phosphoramidate dimer(Example 55).

Scheme XII illustrates the preparation of a thiol intermediate and theuse of that intermediate with an amino nucleoside to form aS-iminomethylene linkage (Example 58). As with the reactions of SchemeXI, a dimeric unit having an active phosphoramidate moiety can beformed. This is exemplified by Examples 59 and 60.

Scheme XIII illustrates the preparation of a nucleoside intermediate andcoupling of that intermediate to a further nucleoside, as exemplified inExample 61, to form a nitrilo-1,2-ethanediyl linkage. This linkage canbe reduced to an imino-1,2-ethanediyl linkage, as exemplified in Example62. Further, in a manner similar to Schemes XI and XII, Scheme XIIIillustrates the preparation of an active phosphoramidate species, asexemplified in Examples 63, 64, and 65.

Scheme XIV illustrates the preparation of a 2' substituted nucleoside,as exemplified in Example 66, and conversion of that 2' substitutednucleoside to a further nucleoside having an active linkage formingmoiety (Example 67). Linkage of this 2' substituted nucleoside to afurther nucleoside (Example 68) is followed by conversion to an activephosphoramidate (Example 69). Substitution of the 2' position in amacromolecule of the invention, as noted above, is useful for theintroduction of other molecules, including the introduction of reportermolecules, RNA cleaving groups, groups for improving the pharmacokineticproperties of an oligonucleotide, and groups for improving thepharmacodynamic properties of an oligonucleotide as well as other groupsincluding but not limited to O, S and NH alkyl, aralkyl, aryl,heteroaryl, alkenyl, alkynyl and ¹⁴ C containing derivatives of thesegroups, F, Cl, Br, CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃,NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino and substituted silyl.

Further illustrated in Scheme XIV is the preparation of a carbocyclicnucleoside (Example 70), joining of that carbocyclic nucleoside with afurther nucleoside via a linkage of the invention (Example 71), andformation of an active phosphoramidate (Example 76). A further sequenceof reactions are also illustrated in Scheme XIV, wherein a carbocyclicnucleoside is derivatized at its 2' positions (Example 73) and convertedto a further nucleoside (Example 74). As with the other reaction of thisscheme, a dimer is first formed (Example 75), and then derivatized withan active phosphoramidate (Example 76). The dimers of this scheme havinga 3' phosphoramidite moiety are used as in Schemes XII, XII and XIII tolink the oligonucleosides of the invention to other nucleosides via aphosphodiester, phosphorothioate or other similar phosphate basedlinkage.

Scheme XV illustrates a further carbocyclic containing, dimericnucleoside. Internucleoside conversion is exemplified in Examples 77 and78, and formation of a dimeric structure is exemplified in Example 79.The dimeric structure of Scheme XV shows a carbocyclic nucleoside as the5' nucleoside of the dimer, while Scheme XIV shows a carbocyclicnucleoside as the 3' nucleoside of the dimer. Use of carbocyclicnucleosides for both nucleoside intermediates, in the manner asdescribed for other of the reaction schemes, results in a dimer having acarbocyclic nucleoside at both the 3' and 5' locations.

Scheme XVI illustrates generic structures that are prepared from thenucleosides and oligonucleoside of the previous schemes. Exemplarymacromolecules of the invention are described for both solid support andsolution phase synthesis in Example 81. ##STR3##

The compounds of this invention can be used in diagnostics and asresearch reagents and kits.

It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation or activity is to be modulated. The targetingportion of the composition to be employed is, thus, selected to becomplementary to the preselected portion of DNA or RNA, that is to be ancomplementary oligonucleotide for that portion.

In accordance with one preferred embodiment of this invention, thecompounds of the invention hybridize to HIV mRNA encoding the tatprotein, or to the TAR region of HIV mRNA. In another preferredembodiment, the compounds mimic the secondary structure of the TARregion of HIV mRNA, and by doing so bind the tat protein. Otherpreferred compounds complementary sequences for herpes, papilloma andother viruses.

This invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes. Such selective, strong bindingis accomplished by interacting such RNA or DNA with compositions of theinvention which are resistant to degradative nucleases and whichhybridize more strongly and with greater fidelity than knownoligonucleotides or oligonucleotide analogs.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting,wherein parts and percents are by weight unless otherwise indicated. ForNMR analysis of dimers and other higher oligonucleosides, monomericunits are numbered (e.g., T₁, T₂) from the 5' terminus towards the 3'terminus nucleoside. Thus, the 5' nucleoside of a T--T dimer is T₁ andthe 3' nucleoside is T₂.

EXAMPLE 1 Synthesis of 5'-deoxy-5'-hydrazino nucleosides

(a) 5'-Deoxy-5'-hydrazinothymidine hydrochloride

To provide 5'-benzylcarbazyl-5'-doxythymidine, 5'-O-tosylthymidine,[Nucleosides & Nucleotides 1990, 9, 89] (1.98 g, 5 mmol),benzylcarbazide (4.15 g, 25 mmol), activated molecular sieves (3A, 2 g),and anhydrous dimethylacetamide (100 ml) were stirred together withexclusion of moisture at 110° C. (bath temperature) for 16 hours. Theproducts were cooled and concentrated under reduced pressure (bathtemperature <50° C.). The residue was purified on a silica gel column(5×45 cm) with CH₂ CL₂ /MeOH (9:1, v/v) as the solvent. The homogeneousfractions were pooled, evaporated to dryness and the foam recrystallizedfrom EtOH to yield 0.7 g (36%) of 5'-benzylcarbazyl-5'-deoxythymidine;mp 201° C.; ¹ H NMR (Me₂ SO--d₆)δ1.79 (s, 3, CH₃), 2.00-2.18 (m, 2,C_(2') CH₂), 2.95 (t, 2, C_(5') CH₂), 3.75 (m, 1, C_(4') H), 4.18 (m, 1,C_(3') H), 4.7 (brs, 1, O'₂ NH), 5.03 (s, 2, PhCH₂), 5.2 (d, 1, C_(3')H), 6.16 (t, 1, C_(1') H), 7.2-7.4 (m, 5, C₆ H₅), 7.6 (s, 1, C₆ H), 8.7(brs, 1, CH₂ NH), 11.2 (brs, 1, C₃ NH).

To provide the hydrochloride salt of 5'-deoxy-5'-hydrazinothymidine as ahygroscopic powder, a mixture of the above carbazate (0.78 g, 2 mmol)and palladium on charcoal (10%, 150 mg) in anhydrous MeOH/HCl (30 ml,2%, HCl by weight) was stirred under an atmosphere of hydrogen at roomtemperature of 1.5 hours. The methanolic solution was filtered throughCelite to remove the catalyst. The filter cake was washed with EtOH(2×25 ml). The filtrate was concentrated under vacuum and the residuewas dried overnight to remove traces of HCl. The yellow residue wasdissolved in methanol (3 ml) and added dropwise to a rapidly stirredsolution of ethyl acetate (150 ml). The filtered precipitate was washedwith ethyl acetate (3×100 ml) and the pale yellow solid was dried undervacuum to yield 0.51 g (88%) of 5'-deoxy-5'-hydrazinothymidinehydrochloride (hygroscopic powder); ¹ H NMR (Me₂ SO--d₆) δ 1.81 (s, 3,CH₃), 2.02-2.22 (m, 2, C_(2') CH₂), 3.2 (m, 2, C_(5') CH₂), 3.8, (m, 1,C_(4') H), 4.2 (m, 1, C_(3') H), 6.17 (t, 1, C_(1') H), 7.54 (s, 1, C₆H), 11.18 (brs, 1, C₃ NH), the hydrazino and 3'-OH were masked by H₂ O.

EXAMPLE 2 Synthesis of5'-O-trityl-1-[2,3-dideoxy-3-C-(formyl)-β-D-erythro-pentofuranosyl]-thymineand -uracil Method A 3'-C-Cyano-3'-deoxy-5'-O-tritylthymidine

The following preparation should to be performed under a hood and allprecautions taken not to inhale any of reagent fumes. A suspension of3'-deoxy-3'-iodo-5'-O-tritylthymidine (Verheyden, et al., J. Org. Chem.1970, 35, 2868) (60 g, 0.1 mol), hexamethylditin (36 g, 22.7 ml, 0.11mol), t-butylisocyanide (166 g, 225 ml, 2 mol), and AIBN (1.6 g, 10mmol) in toluene (freshly distilled over Na/benzophenone, 2 lt) wasthoroughly deoxygenated by bubbling argon through the reaction mixturefor 30 min. and then heated at 80° C. for 13 h. The reaction mixture wascooled at 60° C. and AIBN (1.6 g, 10 mmol) was added and heatingcontinued for 24 h. During this period addition of AIBN was repeated for3 times in an identical manner. The reaction mixture was cooled to roomtemperature and transferred on the top of a prepacked silica gel column(1.5 kg, in hexanes) and eluted with hexanes: Et₂ O (100% hexanes→100%Et₂ O with a 10% gradient change each time using 1 lt of eluent). Mostof the impurities were removed during the gradient elution as non-polarcompounds. Final elution with Et₂ O (2 lt), pooling and evaporation ofappropriate fractions gave two compounds in the order these werecollected. (i) 12.93 g (25%) of 3'-C-Cyano-3'-deoxy-5'-O-tritylthymidineas white powder (crystallized from toluene/Et₂ O, mp 153°-157° C.); ¹ HNMR (CDCl₃) δ 8.83 (s, 1, NH), 7.04-7.4 (m, 18.5, TrH, C₆ H, and 0.5 ArHfrom toluene), 6.10 (dd, 1, H_(1'), J_(1'),2' =4.1 Hz, J_(1'),2" =7.1Hz), 4.20 (m, 1 , H_(4'), J_(3'),4' =8.4 Hz, J_(4'),5' =2.8 Hz),3.33-3.60 (m, 3, H_(5'), 5", 3'), 2.68 (m, 1, H_(2'), J_(2'), 2" =13.8Hz), 2.52 (m, 1, H_(2")), 2.28 (s, 1.5, 0.5 CH₃ from toluene), and 1.50(s, 3, CH₃).

Anal. Calcd. for C₃₀ H₂₇ N₃ O₄.sup.. 0.5 C₇ H₈ (toluene fromcrystallization); C, 74.56; H, 5.79; N, 7.78. Found: C, 74.27; H, 5.78;N, 7.66.

The reaction mixture also gave 4.82 g, (10%) of1-(3'-C-Cyano-2',3'-dideoxy-5'-O-trityl-β-D-threopentofuranosyl)thymine;¹ H NMR (CDCl₃) δ 8.72 (s, 1, NH), 7.03-7.44 (m, 18.5, TrH, C₆ H, and0.5 ArH from toluene), 6.13 (pseudo t, 1, H_(1'), J_(1'), 2' =6.7 Hz,J_(1'),2" =5.7 Hz), 4.09 (m, 1, H_(4'), J_(3'),4' =6.7 Hz, J_(4'),5'=4.9 Hz), 3.56 (m, 2, H_(5'),5", H_(2'), J_(2'),2" =14 Hz), 2.28 (s,1.5, CH₃ from toluene) and 1.60 (s, 3, CH₃).

Anal. Calcd. for C₃₀ H₂₇ N₃ O₄.sup.. 0.5 C₇ H₈ (toluene fromcrystallization); C, 74.56; H, 5.79; N, 7.78. Found: C, 74.10; H, 5.74;N, 7.52.

Epimerization:

To a suspension of1-(3'-C-Cyano-2',3'-di-deoxy-5'-O-trityl-β-D-threo-pentofuranosyl)thymine(0.30 g, 0.61 mmol) in methanol (20 ml) was added dropwise a 1N solutionof NaOMe until the pH of solution reached ≈9. The resulting mixture washeated to reflux for 20 h. The solution was cooled (0° C.) andneutralized with 1N HCl/MeOH and evaporated under reduced pressure. Theresidue was purified as described above to furnish 0.185 g) (62%) of3'-C-cyano-3'-deoxy-5'-O-tritylthymidine. (A synthesis for3'-deoxy-3'-C-cyano-5'-O-tritylthymine was reported in TetrahedronLetters 1988, 29, 2995. This report suggested3'-deoxy-3'-C-cyano-5'-O-tritylthymine is formed as a single product,however, we found a mixture of threo- and erythro-3'-cyano isomers areproduced. (see, Bankston, et al., J. Het. Chem. 1992, 29, 1405. By theabove epimerization, the xylo component of this mixture is converted tothe compound of interest, 3'-deoxy-3'-C-cyano-5'-O-tritylthymine.)

3'-Deoxy-3'-C-formyl-5'-O-tritylthymine

DIBAL-H (1M in toluene, 50 ml, in 5 portions over a period of 5 h) wasadded to a stirred solution of 3'-C-cyano-3'-deoxy-5'-O-tritylthymidine(9.92 g, 20 mmol) in dry THF (10 ml) under argon at 0° C. The solutionwas stirred at room temperature for 1 h and cooled again to 0° C. MeOH(25 ml) was added dropwise to the cold solution while stirring and aftercomplete addition the solution was brought to room temperature. Asaturated aqueous Na₂ SO₄ solution (11 ml) was added to the reactionmixture and stirred for 12 h. Powdered anhydrous Na₂ SO₄ (30 g) wasadded to the reaction mixture and suspension was stirred for 30 min. Thesuspension was filtered and residue was thoroughly washed with MeOH:CH₂Cl₂ (1:9 v/v) until all of the product was washed off. The filtrateswere combined and concentrated under vacuum, to furnish a gummy residue.The residue was purified by silica gel chromatography using CH₂ Cl₂:MeOH (100% CH₂ Cl₂ →9:1, v/v) for elution to obtain 5.45 g (55%) of3'-deoxy-3'-C-formyl-5'-O-tritylthymine as a white foam. ¹ H NMR (CDCl₃)δ 9.61 (d, 1, CHO, J_(3'),3" =1.5 Hz), 8.44 (s, 1, NH), 7.46 (s, 1, C₆H), 7.17-7.45 (m, 15, TrH), 6.04 (pseudo t, 1, H_(1'), J_(1'), 2' =5.3Hz, J_(1'),2" =6.6 Hz), 4.31 (m, 1, H_(4'), J_(4'), 5' =3.3 Hz, J_(3'),4' =7 Hz), 3.28-3.55 (m, 3, H_(5'), 5", 3'), 2.69 (m, 1, H_(2')), 2.28(m, 1, H_(2") ), 1.48 (s, 3, CH₃).

Anal. Calcd. for C₃₀ H₂₈ N₂ O₅.sup.. H₂ O: C, 70.03; H, 5.88; N, 5.44.Found: C, 70.40; H, 6.00; N, 5.33.

1-[3-Deoxy-3-C-(formyl)-5-O-trityl-β-D-erythropentofuranosyl]uracil

To a stirred solution of 3'-cyano-2',3'-dideoxy-5'-O-trityluridine (0.96g, 2 mmol), (prepared in a manner equivalent to that of the thymidineanalog above) in dry THF (20 ml) under argon, was added a solution ofDIBAL-H in toluene (Aldrich) (1M, 4 ml) at -10° C. over a period of 10min. After 30 mins the reaction was quenched with MeOH (5 ml) at -10° C.The mixture was further stirred at ambient temperature for 30 mins anddiluted with CH₂ Cl₂ (25 ml) before concentrating under vacuum. Thisprocess was repeated with CH₂ Cl₂ (3×25 ml) in order to remove theresidual THF. The residue was purified by flash chromatography on silicagel (25 g). Elution with CH₂ Cl₂ (9:1, v/v) and crystallization from CH₂Cl₂ /MeOH gave 5'-O-trityl-3'-C-formyl-2',3'-dideoxyuridine (0.53 g,53%); mp 100° C.; ¹ H NMR (CDCl₃) δ 2.25-2.8 (m, 2, CH₂), 3.4 (m, 1,C_(3') H), 3.45-3.6 (m, 2, C_(5'CH) ₂), 4.37 (m, 1, C_(4') H), 5.4 (d,1, C₅ H), 6.1 (m, 1, C_(1') H), 7.2-7.4 (m, 15, C₆ H₅), 7.81 (d, 1, C₆H), 7.95 (br s, 1, NH), 9.61 (s, 1, HC═O).

Method B1-[3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythropentofuranosyl]thymine

1-Methyl-5-O-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-(formyl)-D-erythro-pentofuranosewas obtained as an oil in 90% yield using the DIBAL-H reduction of1-methyl-5-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-cyano-D-erythro-pentofuranoseas described in Tetrahedron 1900, 44, 625. The 3-C-formyl group isderivatized to the oxime with methoxyamine. The oxime blockedintermediate was glycosylated with silyated thymine to give an α and βmixture of the title compound. After deblocking, the β anomer comparesto that prepared via method A.

Method C1-[3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythropentofuranosyl]-uracil and-thymine

A mixture of 3'-deoxy-3'-iodo-5'-O-tritylthymidine (0.59 g, 4 mmol),tris(trimethylsilyl) silane (2.87 g, 1.2 mmol), AIBN (12 mg, 0.072mmol), and toluene (20 ml) were mixed in a glass container and saturatedwith argon (bubbling at room temperature). The glass vessel was insertedinto a stainless steel pressure reactor, and pressurized with carbonmonoxide (80 psi), closed and heated (90° C., bath) for 26 hrs. Thereaction mixture was cooled (0° C.) and the CO was allowed to escapecarefully (under the fume hood). The product was purified by flashcolumn chromatography on silica gel (20 g). Elution with EtOAc: Hexanes(2:1, v/v) and pooling the appropriate fractions furnished 0.30 g (61%)of the title compound as a foam.

A radical carbonylation of 2',3'-dideoxy-3'-iodo-5'-trityluridine in asimilar manner gives the 3'-C-formyl uridine derivative.

EXAMPLE 3 Synthesis of methylenehydrazone linked (3'--CH═NH--NH--CH₂--5'), methylenehydrazine linked (3'--CH₂ --NH--NH--CH₂ --5') andmethylene(dimethylhydrazo) linked (3'--CH₂ --N(CH₃)--N(CH₃)--CH₂ --5')dinucleosides3'-De(oxyphosphinico)-3'-[methylene(hydrazone)]-5'-O-tritylthymidylyl-(3'.fwdarw.5')-5'-deoxythymidine

A mixture of 3'-deoxy-3'-C-formyl-5'-O-tritylthymidine, 0.645 g, 1.30mmol), 5'-deoxy-5'-hydrazinothymidine hydrochloride (0.397 g, 1.36 mmol)in dry CH₂ Cl₂ /MeOH/AcOH (20 ml/10 ml/0.5 ml) was stirred for 30 min atroom temperature. The solvent was evaporated under vacuum and thehydrazone intermediate was analyzed by ¹ H NMR (DMSO-d₆) δ 1.1 (br s, 2NH), 8.3 (s, 1, C═N--NH), 7.5-7.74 (m, 17, Tr H, 2C₆ H), 6.8 (1d, 1t, 1,HC═N, two isomers), 6.0-6.1 (2m, 2, H₁,), 5.30 (br t, 1, OH), 3.8-4.2(3m, 3, H_(3'), 2 H_(4')), 3.0-3.3 (m, 5, 2H_(5'), 5", H_(3')), 2.0-2.4(m, 4, 2H_(2'), 2), 1.5 and 1.7 (2s, 6, 2 CH₃).

3'-De(oxyphosphinico)-3'-[methylene(dimethylhydrazo)]-5'-O-tritylthymidylyl-(3'→5')-5'-deoxythymidine

The above hydrazone dimer was dissolved in AcOH (10 ml) and to this wasadded small portions of NaBH₃ CN (4×0.12 g, 7.74 mmol) while stirring atroom temperature for 30 min. The solution was stirred for an additional15 min before the addition of aqueous HCHO (20%, 3.9 ml, 26 mmol, NaBH₃CN (3.9 mmol), and AcOH (10 ml). The suspension was further stirred for15 min. and solution evaporated under vacuum. The residue wascoevaporated with MeOH (3×25 ml) to give the methylenehydrazo dimer; ¹ HNMR (CDCl₃) δ 6.8-7.8 (m, 15, TrH, 2 C₆ H), 6.12 (m, 2, 2H_(1')), 4.20((m, 1, T2 H_(3')), 4.05 (m, 1, T2 H_(4')), 3.89 (m, 1, T1 H_(4')), 3.80(s, 6, 2 OCH₃), 3.21-3.53 (m, 2, T1 H_(5'),5"), 2.11-2.75 (m, 10, T2H_(5'5") H, T1 H_(3"), T1 H_(3'), T1 T2 H_(2'2")), 2.26 (s, 6, 2N--CH₃),1.88 and 1.49 (2s, 6, 2 CH₃), and other protons.

3'-De(oxyphosphinico)-3'-[methylene(dimethylhydrazo)]-thymidylyl-(3'.fwdarw.5')-5'-deoxythymidine

The above hydrazine dimer was then stirred with 37% aqueous HCl (1 ml)in MeOH (25 ml) at room temperature for 24 h. The resulting mixture wasneutralized with NH₄ OH (pH≈8) and evaporated to dryness. The residuewas purified by reverse phase HPCL (supelcosil LC18, 5 m, H₂ O: CH₃ CNgradient) to furnish 0.61 g of the title methylene(dimethylhydrazine)linked dimer (89%). ¹ H NMR (90° C., DMSO-d₆ +1 drop of D₂ O) δ 7.66 and7.43 (2s, 2, 2 C6H), 6.02 (pseudo t, 1, T2 H_(1'), J_(1'),2' =7.2 Hz,J_(1'),2" =7.7 Hz), 5.96 (pseudo t, 1, T1 H_(1'), J_(1'),2' =5.6 Hz,J_(1'),2" =6.2 Hz), 4.12 (m, 1, T2 H_(3')), 3.90 (m, 1, T2 H_(4')), 3.71(m, 1, T1 H_(4')), 3.61 (m, 2, T1 H_(5'),5"), 2.4-2.8 (m, 5, T2H_(5'),5", T1 H_(3"), T1 H_(3')), 2.29 (2s, 6, 2 N--CH₃), 2.12 (m, 4,2H_(2'),2"), 1.76 and 1.74 (2s, 6, 2 CH₃).

Anal. Calcd. for C₂₃ H₃₄ N₆ O₈, H₂ O: C, 51.10, H, 6.71; N, 15.54.Found: C, 51.05; H, 6.68; N, 15.54. MS FAB m/z 523 (M+H)⁺.

EXAMPLE 4 Synthesis of methylene(dimethylhydrazine) linked (3'-CH₂-N(CH₃)-N(CH₃)-CH₂ -5')5'-dimethoxytrityl-3'-β-cyano-ethoxydiisopropylphosphoramiditedinucleosides3'-De(oxyphosphinico)-3'-[methylene(dimethylhydrazo)]-thymidylyl-5'-O-(dimethoxytriphenylmethyl)-(3'→5')-3'-O-(β-cyanoethyl-N-diisopropylaminophosphiryl)thymidine

The methylene(dimethylhydrazine) dimer of Example 3 wasdimethyoxytritylated following the standard procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish a homogenous foam ¹ H NMR (CDCl₃) δ 6.8-7.8 (m,20, DMTr, 2H₆), 6.12 (m, 2, 2H_(1')), 4.20 (m, 1, T₂ H_(3')), 4.05 (m,1, T₂ H_(4')), 3.89 (m, 1, T₁ H_(4')), 3.80 (s, 6, 2 OCH₃ of DMTr),3.21-3.53 (m, 2, T₁ H_(5'5")), 2.11-2.75 (m, 9, T₁ H_(5'5"), H_(3"), T₁H_(3'), 2H_(2'2")), 2.26 (2s, 6, 2 N--CH₃) and 1.88 and 1.49 (2s, 2, C₅CH₃) ] which on phosphitylation via the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, provided a 65% yield of the title compound. ¹ H NMR (CDCl₃)δ 6.14 (m, 1, T2 H_(1')), 6.01 (m, 1, T1 H_(1')), 3.80 (s, 6, 2 O CH₃),2.23 (m, 6, 2 N--CH₃), 1.78 and 1.45 (2s, 6, 2 CH₃), and other protons.³¹ P NMR (CDCl₃) δ 149.43 and 148.85 ppm.

EXAMPLE 5 Synthesis of intermittent methylene(dimethylhydrazine) (3'-CH₂-NCH₃ -NCH₃ -CH₂ -5') linked oligonucleosides

CPG-bound thymidine (or any other nucleoside that is to become the3'-terminal base) was placed in an Applied Biosystems, Inc. (ABI) column(250 mg, 10 micromoles of bound nucleoside) and attached to an ABI 380Bautomated DNA Synthesizer. The standard, automated (computer controlled)steps utilizing phosphoramidite chemistries are employed to place themethylenehydrazine thymidine dimer into the sequence at any desiredlocation.

EXAMPLE 6 Synthesis of 5'-O-phthalimido nucleosides5'-O-Phthalimidothymidine

To a stirred solution of thymidine (24.22 g, 0.1 mol),N-hydroxyphthalimide (21.75 g, 0.13 mol), triphenylphosphine (34 g, 0.13mol) in dry DMF (400 ml) was added diisopropylazodicarboxylate (30 ml,0.15 mol) over a period of 3 h at 0° C. After complete addition thereaction mixture was warmed up to room temperature and stirred for 12 h.The solution was concentrated under vacuum (0.1 mm, <40° C.) to furnishan orange-red residue. The residual gum was washed several times withEt₂ O and washing were discarded. The semi-solid residue was suspendedin EtOH (500 ml) and heated (90° C.) to dissolve the product. On cooling30.98 g (80%) of 5'-O-phthalimidothymidine was collected in 3-crops aswhite crystalline material, mp 233°-235° C. (decomp.); ¹ H NMR (DMSO-d₆)δ 11.29 (s, 1, NH), 7.85 (m, 4, ArH), 7.58 (s, 1, C₆ H), 6.20 (t, 1,H_(1'), J_(1'),2' =7.8 Hz, J_(1'),2" =6.5 Hz), 5.48 (d, 1, OH_(3')),4.36 (m, 3, H_(4'),5',5"), 4.08 (m, 1, H_(3')), 2.09-2.13 (m, 2, H_(2'),2"), and 1.79 (s, 3, CH₃).

Anal. Calcd. for C₁₈ H₁₇ O₇ N₃.0.7 H₂ O: C, 54.05; H, 4.64; N, 10.51.Found: C, 53.81; H, 4.25; N, 10.39.

2'-deoxy-5'-O-phthalimidouridine

An analogous reaction on 2'-deoxyuridine gave the corresponding2'-deoxy-5'-O-phthalimidouridine; mp 241°-242° C.

EXAMPLE 7 Synthesis of5'-O-phthalimido-3'-O-(t-butyldiphenylsilyl)thymidine and2'-deoxy-5'-O-phthalimido-3'-O-(t-butyldiphenylsilyl)uridine3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine

A mixture of 5'-O-phthalimidothymidine (8.54 g, 22 mmol),t-butyldiphenylsilylchloride (6.9 ml, 26.5 mmol), imidazole (3.9 g, 57.3mmol) and dry DMF (130 ml) was stirred at room temperature for 16 hunder argon. The reaction mixture was poured into ice-water (600 ml) andthe solution was extracted with CH₂ Cl₂ (2×400 ml). The organic layerwas washed with water (2×250 ml) and dried (MgSO₄). The CH₂ Cl₂ layerwas concentrated to furnish a gummy residue which on purification bysilica gel chromatography (eluted with EtOAc:Hexanes; 1:1, v/v)furnished 12.65 g (92%) of3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine as crystallinematerial (mp 172°-173.5° C.). ¹ H NMR (DMSO-d₆) δ 11.31 (s, 1, NH), 7.83(m, 4, ArH), 7.59 (m, 4, TBDPhH), 7.51 (s, 1, C₆ H), 7.37-7.45 (m, 6,TBDPhH), 6.30 (dd, 1, H_(1'), J_(1'), 2' =8.8 Hz, J_(1'),2" =5.6 Hz),4.55 (m, 1, H_(4')), 4.15 (m, 1, H_(3')), 3.94-4.04 (m, 2, H_(5'),5"),2.06-2.13 (m, 2, H_(2'),2"), 1.97 (s, 3, CH₃), 1.03 (s, 9, C(CH₃)₃).

Anal. Calcd. for C₃₄ H₃₅ O₇ N₃ Si: C, 65.26; H, 5.64; N, 6.71. Found: C,65.00; H, 5.60; N, 6.42.

3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidouridine

An analogous reaction of 2'-deoxy-5'-O-phthalimidouridine will give thecorresponding3'-O-(t-butyldiphenylsilyl)-2'-deoxy-5'-O-phthalimidouridine.

EXAMPLE 8 Synthesis of 5'-O-amino nucleoside5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine

To a stirred solution of3'-O-(t-butyldiphenylsilyl)-5'-O-phthalimidothymidine (10 g, 16 mmol),in dry CH₂ Cl₂ (100 ml) was added methylhydrazine (1.3 ml, 24 mmol)under argon at room temperature and solution stirred for 12 h. Thesolution was cooled (0° C.) and filtered. The white residue was washedwith CH₂ Cl₂ (2×25 ml) and combined filtrates were evaporated to furnishgummy residue. The residue on purification by silica gel columnchromatography (elution with CH₂ Cl₂ :MeOH, 98:2, v/v) furnished 7.03 g(89%) of 5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine thatcrystallized from CH₂ Cl₂ /MeOH mp 141°-143° C. ¹ H NMR (DMSO-d₆) δ11.29 (s, 1, NH), 7.42-7.62 (m, 11, TBDPhH, C₆ H), 6.25 (dd, 1H_(1'),J_(1'),2' =8.4 Hz, J_(1'),2" 6.3 Hz), 6.02 (s, 2, NH₂), 4.35 (m, 1,H_(4')), 4.04 (m, 1, H_(3')), 3.34-3.51 (m, 2, H_(5'),5"), 2.04 (m, 2,H_(2'), 2"), 1.73 (s, 3, CH₃), 1.03 (s, 9, C(CH₃)₃).

Anal. Calcd. for C₂₆ H₃₃ O₅ N₃ Si: C, 63.00; H, 6.71; N, 8.48. Found: C,62.85; H, 6.67; N, 8.32.

EXAMPLE 9 Synthesis of (3'--CH═N--O--CH₂ --5') linked oligonucleoside(an oxime linked dimer)3'-De(oxyphosphinico)-3'-(methylidynenitrilo)-thymidylyl-(3'→5')-thymidine

A mixture of 3'-deoxy-3'-C-formyl-5'-O-tritylthymine (0.99 g, 2 mmol),5'-amino-3'-O-(t-butyldiphenylsilyl) thymidine (0.99 g, 2 mmol) and AcOHtemperature. The solvent was evaporated under vacuum and the crudeblocked3'-de(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-3'-(t-butyldiphenylsilyl)thymidineproduct was dissolved in THF (20 ml). A THF solution of nBu₄ NF (1M, 5ml) was added to the stirred reaction mixture at room temperature. After1 h solution was purified by silica gel chromatography (elution with CH₂Cl₂ :MeOH, 99:4, v/v) to furnish 3'-deblocked dimer. The dimer wasdissolved in anhydrous MeOH (50 ml) and to this a MeOH/HCl solution(0.14M, 2.5 ml) was added. The reaction mixture was stirred at roomtemperature for 15 h. Anhydrous pyridine (10 ml) was added to the abovesolution and solvents were evaporated to dryness to furnish crude oximedimer. The crude product was purified by silica gel chromatography(elution with CH₂ Cl₂ :MeOH; 92:8, v/v) to furnish the title compound,3'-De(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-thymidine,(0.87 g, 89%) as a mixture of E/Z isomers. The two geometrical isomerswere separated by reverse phase HPLC (Supelcosil LC18, 5μ, H₂ O:CH₃ CNgradient). (Z-isomer of title compound) ¹ H NMR (DMSO-d₆) δ 11.28 (br s,2, 2NH), 7.39 and 7.78 (2s, 2, 2C6H), 6.92 (d, 1, T1 H_(3"), J_(3'), 3"6.7 Hz), 6.15 (pseudo t, 1, T2 H_(1'), J_(1'),2' =7.8 Hz, J_(1'),2" =6.3Hz), 6.04 (dd, 1, T1 H_(1'), J_(1'),2' =7.1 Hz, J_(1'),2" =6.3 Hz), 5.34(d, 1, T2 OH), 5.12 (t, 1, T₁ OH), 4.11-4.25 (m, 3, T2 H_(5'5"), T1H_(3')). 3.96 (m, 1, T2 H_(4')), 3.90 (m, 1, T1 H_(4')), 3.49-3.69 (m,3, T1 H_(5'),5", T1 H_(3')), 2.06-2.31 (m, 4, T1 H_(2'),2", T2H_(2'),2"), 1.73 (s, 6, 2CH₃).

Anal. Calcd. for C₂₁ H₂₇ N₅ O₉.H₂ O: C, 49.31; H, 5.72; N, 13.69. Found:C, 49.32; 5.57; N, 13.59. (E-isomer of the title compound) ¹ H NMR(DMSO-d₆) δ 11.3 (2 br s, 2, 2NH), 7.81 (s, 1, C₆ H), 7.52 (d, 1, T1H_(3"), J_(3'),3" =6.7 Hz), 7.45 (s, 1, C₆ H), 6.10 (pseudo t, 1, T2H_(1'), J_(1'),2' =7.6 Hz, J_(1'),2" =6.4 Hz), 6.04 (dd, 1T1 H_(1'),J_(1'),2' =7.3 Hz, J_(1'),2" =3.4 Hz), 5.36 (d, 1, T2 OH), 5.16 (t, 1,T1 OH), 4.07-4.22 (m, 3, T2 H_(3') ,5',5"), 3.91 (m, 2, T1 T2 H_(4')),3.50-3.73 (m, 2, T1 H_(5'),5"), 3.12 (m, 1, T1 H_(3')), 2.05-2.44 (m, 4,T1 T2 H_(2'),2"), and 1.76 (s, 6, 2CH₃). MS FAB: M/z 494 (M+H)⁺.

EXAMPLE 10 Synthesis of phosphoramidate containing (3'--CH═N--O--CH₂--5') linked oligonucleoside3'-De(oxyphosphinico)-3'-(methylidynenitrilo)-5'-O-(dimethyoxytriphenylmethyl)-thymidylyl-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The isomeric dimer of Example 9 was further dimethyoxytrityled at thehydroxyl group of the 5' terminus nucleoside followed by conversion toits 3'-O-β-cyanoethyldiisoprpylphosphoramidite derivative at thehydroxyl group at the 3' terminus nucleoside of the dimer following theprocedure described in Oligonucleotide Synthesis: a practical approach,Ed. M. J. Gait, IRL Press, 1984, to yield the title compound. ¹ H NMR(CDCl₃) δ 8.77 (br s, 2, 2NH), 7.68 (s, 0.77, T1 C₆ H E-isomer), 7.59(s, 0.23, T1 C₆ H, E-isomer), 6.3 (ps t, 1, T2 CH_(1')), 6.14 (m, 0.77,T1 CH_(1') E-isomer), 6.08 (m, 0.23, T₁ CH_(1') Z-isomer), 1.80 and 1.50(2S, 6, 2 CH₃) and other protons. ³¹ P NMR (CDCl₃) 150.77 and 150.38(Z-isomer); 150.57 and 150.38 (E-isomer).

The protected dimer can be conveniently stored and used for couplingutilizing an automated DNA synthesizer (ABI 380B) as and when requiredfor specific incorporation into oligomers. Further as per furtherexamples of the specification, the oxime linked dimer is reduced to adimer bearing a corresponding hydroxylamine linkage and this in turn canbe alkylated to a hydroxylmethylamine or other hydroxyalkylaminelinkage.

EXAMPLE 11 Synthesis of (3'--CH₂ --NH--O--CH₂ --5') linkedoligonucleoside3'-De(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-thymidine

To a stirred solution of blocked dimer3'-de(oxyphosphinico)-3'-(methylidynenitrilo)thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine(0.49 g, 1 mmol) in glacial AcOH (5 ml) was added NaBH₃ CN (0.19 g, 3mmol) in 3-portions under argon at room temperature. The suspension wasstirred for 1 h until bubbling of solution ceased. Additional NaBH₃ CN(0.19 g, 3 mmol) was added in a similar manner and stirring continuedfor 1 h. The AcOH was removed under reduced pressure to furnish3'-de(oxyphosphinico-3'-(methyleneimino)-thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine.Deblocking of this dimer as described before using nBu₄ NF/THF andHCl/MeOH furnished the title compound,3'-de(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-thymidine,(0.44 g, 90%) as white powder. This dimer was further purified by HPLC(as described for the 3'-de(oxyphosphinico)-3'-(methylidyne-nitrilo)thymidylyl-(3'→5')-thymidine dimer of Example 9) to obtain ananalytically pure sample. ¹ H NMR (DMSO-d₆) δ 11.23 (br s, 2, 2NH), 7.83and 7.49 (2s, 2, 2C₆ H), 6.82 (t, 1, NHO), 6.14 (pseudo t, 1, T2H_(1'),J_(1'),2' =7.6 Hz, J_(1'),2" =6.5 Hz), 5.96 (dd, 1, T_(b) H_(1'),J_(1'),2' =6.9 Hz, J_(1'),2" =4.3 Hz), 5.28 (s, 1, T2 OH), 5.08 (s, 1,T1 OH), 4.18 (m, 1, T2 H_(3')), 3.89 (m, 1, T1 H_(4')), 3.54-3.78 (m, 5,T1 T2 H_(5'),5", T2 H_(4')), 2.76 -2.94 (m, 2, T1 H_(3")), 2.42 (m, 1,T1 H_(3')), 2.0-2.17 (m, 4, T1, T2 H_(2'),2"), 1.77 and 1.74 (2s, 6, 2CH₃). MS FAB: M/z 496 (M+H)⁺. Anal. Calcd. for C₂₁ H₂₉ N₅ O₉.sup.. H₂ O:C, 49.12; H, 6.09; N, 13.64. Found: C, 48.99; H, 5.96; N, 13.49.

EXAMPLE 12 Synthesis of methylated [3'-CH₂ -N(CH₃)-O-CH₂ -5'] linkedoligonucleoside

3'-De(oxyphosphinico)-3'-[methylene(methylimino)]thymidylyl-(3'→5')thymidine

To a stirred solution of3'-de(oxyphosphinico)-3'-(methyleneimino)thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine dimer (0.99 g, 1 mmol) in glacial AcOH(10 ml) was added aqueous HCHO (20%, 3 ml). The solution was stirred for5 min. at room temperature and to this was added NaBH₃ CN (0.19 g, 3mmol) in 3-portions under argon at room temperature. The addition ofNaBH₃ CN (0.19 g) was repeated once more and solution was furtherstirred for 1 h. The reaction mixture was concentrated to furnish crude3'-de(oxyphosphinico)-3'-[methylene(methylimino)]-thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)thymidine dimer, which on deblocking (nBu₄NF/THF, HCl/MeOH) furnished the title compound,3'-de(oxyphosphinico)-3'-[methylene(methylimino)]thymidylyl-(3'→5')thymidine, (0.44 g, 87%) as white solids. The3'-de(oxyphosphinico)-3'-[methylene(methylimino)]thymidylyl-(3'→5')thymidine dimer was further purified by preparative HPLC furnishing ananalytically pure sample. ¹ H NMR (DMSO-d₆) δ 11.30 and 11.24 (2s, 2,2NH), 7.82 and 7.50 (2s, 2, 2C₆ H), 6.15 (pseudo t, 1, T2 H_(1'),J_(1'),2' =6.3 Hz, J_(1'),2" =7.3 Hz), 6.00 (pseudo t, 1T1 H_(1'),J_(1'), 2' =4.2 Hz, J_(1'),2" =6.1 Hz), 5.31 (m, 1, T2 OH), 5.08 (m,1T1, OH), 4.17 (m, 1, T2 H_(3')), 3.88 (m, 1, T2 H_(4')), 3.57-3.83 (m,5, T1 T2 H_(5'),5", T1 H_(4')), 2.69 (m, 2, T1 H_(3")), 2.57 (s, 3,N-CH₃), 2.50 (m, 1, T1 H_(3')), 2.05-2.14 (m, 4, T1 T2 H_(2'),2"), 1.79and 1.76 (2s, 6, 2 CH₃). MS FAB: M/z 510 (M+H)⁺. Anal. Calcd. for C₂₃H₃₁ N₅ O₉.sup. . H₂ O: C, 50.09; H, 6.31; N, 13.28. Found: C, 50.05; H,6.21, N, 13.08.

EXAMPLE 13 Synthesis of phosphoramidate containing [3'-CH₂ -N(CH₃)-O-CH₂ -5'] linked oligonucleoside

''-De(oxyphosphinico)-3'-[methylene(methylimino)]-5'-O-(dimethoxytriphenylmethyl)thymidylyl-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The 3'-de(oxyphosphinico)-3'-[methylene(methylimino)]thymidylyl-(3'→5')thymidine dimer of Example 12 was tritylated and phosphitylated asdescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, in an overall yield of 82%. The protected dimerwas purified by silica gel column chromatography (CH₂ Cl₂ :MeOH:Et₃ N;9:1:0.1, v/v) and homogenous fractions were pooled and evaporated tofurnish pure3'-de(oxyphosphinico)-3'-[methylene(methylimino)]-thymidylyl-5'-O-(dimethoxytriphenylmethyl)-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidineas a white foam (used as such fir DNA synthesis). The product wasisolated as a mixture of diastereoisomer: ³¹ P NMR (CDCl₃) δ 149.62 and149.11 ppm; ¹ H NMR (CDCl₃) δ 6.22 (pseudo t, 1, T2 H_(1'), J_(1'),2'=J_(1'),2" =6.7 Hz), 6.16 (pseudo t, 1, T1 H_(1'), J=_(1'),2' =J.sub.1',2" =5.8 Hz), 2.58, 2.56 (2s, 3, N-CH₃), 1.82, 1.49 (2s, 6, 2 CH₃),and other protons.

The above protected phosphoramidate bearing dimer can be convenientlystored and used for coupling utilizing an automated DNA synthesizer (ABI380B) as and when required for specific incorporation into oligomers.Other dimers of the inventions, as for example but not limited the abovenoted methylidynenitrilo, i.e., oxime, and methyleneimino, i.e.,aminohydroxy, dimers are converted to their correspondingphosphoramidate derivatives in the same manner as this example andincorporated into oligonucleotide in the standard manner as noted below.An oligomer bearing the oxime linked nucleoside dimer is reduced to anoligomer bearing a corresponding hydroxylamine linked nucleoside dimer.As noted in other examples, reduction can be effected as an CPG boundoligomer or after removal from the CPG.

EXAMPLE 14 Synthesis of intermittent (3'-CH|N-O-CH₂ -5'), i.e., oxime;(3'-CH₂ -NH-O-CH₂ -5'), i.e., aminohydroxy; (3'-CH₂ -N(CH₃)-O-CH₂ -5'),i.e., N-methyl-aminohydroxy; (3'-CH₂ -O-N(CH₃)-CH₂ -5'), i.e.,N-methyl-hydroxyamino; or (3'-CH₂ -N(CH₃)-N(CH₃)-CH₂ -5'), i.e.,N,N'-dimethyl-hydrazino linked oligonucleosides

An appropriate 2'-deoxynucleoside that will become the 3'-terminalnucleoside of an olgionucleoside is bound to a CPG column for use on anABI 380B automated DNA synthesizer. Standard phosphoramidite chemistryprogram steps were employed to place the dimer bearing the(3'-CH|N-O-CH₂ -5'), i.e., oxime; (3'-CH₂ -NH-O-CH₂ -5'), i.e.,aminohydroxy; (3'-CH₂ -N(CH₃)-O-CH₂ -5'), i.e., N-methyl-aminohydroxy;(3'-CH₂ -O-N(CH₃)-CH₂ -5'), i.e., N-methyl-hydroxyamino; or (3'-CH₂-N(CH₃)-N(CH₃)-CH₂ -5'), i.e., N,N'-dimethylhydrazino, linkages into thedesired position or positions of choice within the sequence.

EXAMPLE 15 General and specific NaBH₃ CN reduction for conversion of(3'-CH|N-O-CH₂ -5') linkage to (3'-CH₂ -NH-O-CH₂ -5') Reduction of aDimer

To a solution of a dimer (0.49 g, 1 mmol) in glacial acetic acid (AcOH)(5 ml) was added sodium cyanoborohydride (0.19, 3 mmol) in AcOH (1 ml),under an argon atmosphere at room temperature. The suspension wasstirred for 1 h, and an additional amount of NaBH₃ CN in AcOH (1 ml) wasadded and stirring continued for 1 h. The excess of AcOH was removedunder reduced pressure at room temperature. The residue was coevaporatedwith toluene (2×50 ml) and purified by silica gel (25 g) columnchromatography. Elution with CH₂ CL₂ :MeOH (9:1, v/v) and pooling ofappropriate fractions, followed by evaporation furnished 0.36 g (75%) ofsolid dimer.

Reduction of an Oligonucleoside

CPG-bound oligonucleoside (1 μM), that contains one (or more) backbonemodified linkages is taken off the DNA synthesizer after completion ofits synthesis cycles. A 1.0M NaBH₃ CN solution in THF/AcOH (10 ml, 1:1v/v) is pumped through the CPG-bound material in a standard wayutilizing a syringe technique for 30 min. The column is washed with THF(50 ml), and the reduced oligonucleoside is released from the supportcolumn in a standard way.

Alternative Reduction of an Oligonucleoside

As an alternative to the above reduction, reduction can also beaccomplished after removal from the CPG support. At the completion ofsynthesis the oligonucleoside is removed from the CPG-support bystandard procedures. The 5'-O-trityl-on oligonucleoside is purified byHPLC and then reduced by the NaBH₃ CN/AcOH/THF method as describedabove.

EXAMPLE 16 Synthesis of (3'-CH₂ -N(CH₃)-O-CH₂ -5') linkedoligonucleoside having a 2',3'-didehydro nucleoside as its 5' terminalnucleoside

3'-(de(oxyphosphinico)-2',3'-didehydro-3'-[methylene(methylimino)]thymidylyl-(3'→5')thymidine.

to a stirred solution of1-(5'-O-(MMTr)-β-D-glycero-pentofuran-3'-ulosyl]thymine (0.13 mmol;prepared according to the procedure of T.-C. Wu. et al., Tetrahedron,1989, 45:855, 5'-O-(methyleneamino)-3'-O-(t-butyldiphenylsilyl)thymidine(0.13 mmol; prepared according to the procedure of Debart, et al.,Tetrahedron letters 1992, 33, 2645, ethylene glycol (0.5 mmol), and HMPA(0.5 ml) was added SmI₂ in THF (0.1 mol, 3 ml, 3 mmol) at roomtemperature. The color of SmI₂ fades out as the reaction proceeds toform the desired adduct. After complete disappearance of startingmaterials the reaction mixture is worked-up in the usual way. (Theproduce could be purified by silica column chromatography forcharacterization). The crude mixture of 3'-epimeric adduct is thenalkylated (HCHO/NaCNBH₃ /ACOH) as described in other of these examples.The methylated product is then treated with methylsulfonylchloride inpyridine to obtain a 3'-epimeric mesylate, which on base treatment wouldfurnish the title compound.

EXAMPLE 17 Synthesis of (3'-CH₂ -CH₂ -NH-CH₂ 5') linked oligonucleoside3'-De(oxyphosphinico)-3'-(1,2-ethanediylimino)-thymidylyl-5'-O-(t-butyldimethylsilyl)-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5 '-deoxythymidine

To a stirred solution of aldehyde [2.5 g, 6.5 mmol, freshly preparedaccording to the procedure described by Fiandor, et al., TetrahedronLetts. 1990, 33, 597],5'-amino-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine [3.13 g, 6.5mmol, prepared in two steps via 3'-O-silylation of5'-azido-5'-deoxythymidine in the manner of Hata, et al., J. Chem. Soc.Perkin I1980, 306, and subsequently reduction of the product by themethod of Poopeiko, et al., Syn. Lett. 1991, 342], AcOH (0.39, and 6.5mmol) in dicholoroethane (65 ml) was added followed by NaBH(OAc)₃(2.759, 13.08 mmol) under argon. The suspension was stirred for 3 hoursat room temperature. The reaction mixture was diluted with CH₂ Cl₂ (250ml) and washed with water (2×100 ml). The organic layer was dried(MgSO₄) and concentrated to furnish the crude product as a syrup. Theproduct was purified by silica gel column chromatography to furnish thetitle compound as white foam (3.5 g, 64%). ¹ H NMR (CDCl₃) δ 0.1 [s, 6,Si(CH₃)₂ ]; 0.9 and 1.1 [2s, 18, 2 Si(CH₃)₃ ]; 1.85 and 1.95 (2s, 6, 2CH₃); 2.5 (m, 2, 3"CH₂); 3.7 (m, 2, 5'CH₂); 4.0 (m, 2, 3',4' CH); 4.2(m, 1,3'CH); 6.05 (m, 1, 1'H); 6.28 (t, 1, 1'H); 7.1 and 7.57 (2s, 2,C6H); 7.35-7.7 [2m, 12, Si ArH)₂ ], and other sugar protons.

3'-De(oxyphosphinico)-3'-(1,2-ethanediylimino)-thymidylyl-(3'→5')-5'-deoxythymidine

The protected dimer was deblocked in 81% yield following the standardprocedure using (Bu)₄ NF in THF. The deblocked dimer was purified byHPLC for analysis. ¹ H NMR (DMSO-D₆) δ 1.76 and 1.78 (2s, 6, CH₃);2.0-2.2 (3m, 4, 2'CH₂); 3.15 (m, 2, NCH₂); 3.56 (m, 2, 4'H, 5'CH₂); 4.18(br s, 1, 3'H); 5.17 and 5.22 (2 br s, 2, 2 OH); 5.95 (t, 1, 1'H); 6.1(t, 1, 1'H); 7.6 and 7.85 (2s, 2, 2(C₆ H)); 11.25 (br s, 2 2NH) andother protons.

EXAMPLE 18 Synthesis of Monomer Unit for (3'-CH₂ -O-N|CH-5'), (3'-CH₂-O-NH-CH₂ -5') and (3'-CH₂ -O-N(CH₃)-CH₂ -5') Linkages

1-[3'-Deoxy-3'-C-(hydroxymethyl)-5'-O-(trityl)-β-D-erythro-pentofuranosyl]-thymine

A suspension of NaBH₄ (1.36 g, 9.6 mmol) was added dropwise to a stirredsolution of 3'-C-formyl-5'-O-tritylthymidine in EtOH:H₂ O (22 ml, 3:1,v/v) mixture at room temperature. After 3 h, EtOAc (300 ml) was addedand the organic layer was washed with H₂ O (2×150 ml). The dried (MgSO₄EtOAc extract was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(9:1, v/v), pooling and concentration of appropriate fractions gave thetitle compound (1.13 g, 83%). ¹ H-NMR (CDCl₃) δ 8.29 (br s, 1, NH), 7.59(s, 1, C₆ H) 7.47-7.22 (m, 15, TrH) 6.13 (dd, 1, H_(1'), J_(1'),2' =6.5Hz); 3.98 (m, 1, H_(4')); 3.62 (m, 2, H_(3")), 3.56-3.33 (m, 2, H_(5'),H_(5")), 2.60 (m, 1, H_(3')); 2.33-2.20 (m, 2, H_(2') H_(2')); 1.91 (brs, 1, OH); 1.53 (S, 3, CH₃).

1-[3'-Deoxy-3'-C-[O-(phthalimidohydroxymethyl)]-5'-O-trityl-β-D-erythro-pentofuranosyl]-thymine

Diisopropylazodicarboxylate (0.47 ml, 2.41 mmol) was added to a stirredsolution of 3'-deoxy-3'-C-(hydroxymethyl)-5'-O-trityl-thymidine (0.8 g,1.62 mmol), N-hydroxyphthalimide (0.35 g, 2.15 mmol), triphenylphosphine(0.56 g, 2.15 mmol) in dry THF (10 ml) at room temperature. After 48 h,the products were concentrated and the residue was extracted with CH₂Cl₂ (2×100 ml). The CH₂ Cl₂ extracts were washed with NaHCO₃ (5%, 100ml) and water (100 ml). The dried (MgSO₄) extract was evaporated underreduced pressure and the residue was purified by short-silica gelchromatography. Elution with EtOAC:Hexanes (1:1, v/v), pooling andconcentration of appropriate fractions gave the title compound as whitefoam (0.82 g, 79%). ¹ H-NMR (CDCl₃) δ 8.24 (s, 1, NH); 7.85-7.20 (m, 20,TrH, ArH, C₆ H), 6.20 (m, 1, H₁ ' ), 4.22-4.16 (m, 3, H_(4'), H_(3")),3.63-3.40 (m, 2, H_(5'), H_(5")), 3.02 (m, 1, H_(3')), 2.50-2.43 (m, 2,H₂, H_(2")); 1.51 (s, 3, CH₃). Anal. Calcd. for C₃₈ H₃₃ N₃ O₇. 0.5EtOAc:C, 69.86; H, 5.42, N, 6.11. Found: C, 70.19; H, 5.27; N, 5.75

1-{3'-Deoxy-3'-C-[O-(aminohydroxymethyl)]-5'-O-trityl-β-D-erythro-pentofuranosyl}-thymine

Methylhydrazine (0.12 ml, 2.25 mmol) was added to a stirred solution of3'-deoxy-3'-C-[O-(phthalimidohydroxymethyl)]-5'-O-tritylthymidine (0.77g, 1.2 mmol) in dry CH₂ Cl₂ (9 ml) at room temperature. After 1 h, theprecipitate was filtered and the residue washed with CH₂ Cl₂ (2×10 ml).The combined filtrates were concentrated and the residue was purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH (97:3,v/v), pooling and evaporation of appropriate fractions gave the titlecompound as white powder (0.43 g, 70%). ¹ H-NMR (CDCl₃) δ 8.59 (br s, 1,NH), 7.66 (m, 1, C₆ H), 7.40-7.15 (m, 15, TrH), 6.06 (pseudo t, 1,H_(1')), 5.22 (br s, 2, NH₂), 3.89 (m, 1, H_(4')), 3.65-3.20 (m, 4,H.sub. 5', H_(5"), H_(3")), 2.81 (m, 1, H_(3')), 2.21 -2.13 (m, 2,H_(2'), H_(3")), 1.37 (s, 3, CH₃).

EXAMPLE 19 Synthesis of (3'-CH₂ -O-N|CH-5'), (3'-CH₂ -O-NH-CH₂ -5') and(3'-CH₂ -O-N(CH₃)-CH₂ -5') linked oligonucleosides

3'-De(oxyphosphinico)-3'-[methyleneoxy(methylimino)]thymidylyl-(3'→5')-5'-deoxythymidine

A mixture of1-[4-C-formyl-3-O-(t-butyldiphenylsilyl)-β-D-erythro-pentofuranosyl)thymine[1 mmol, prepared according to the procedure of Nucleosides andNucleotides 1990, 9, 533],3'-deoxy-3'-C-[(O-(aminohydroxymethyl)]-5'-O-tritylthymidine (1 mmol),AcOH (0.1 ml), and dry CH₂ Cl₂ (25 ml) was stirred at room temperaturefor 1 h. The solvent was evaporated and the residue was dissolved inglacial AcOH (5 ml). NaBH₃ CN (3 mmol) was added to the stirred AcOHreaction mixture. After 1 h, an additional amount of NaBH₃ CN (3 mmol)was added and the mixture stirred for 1 h. The reaction was concentratedunder vacuum and the residue was purified by silica gel columnchromatography to furnish 5'-O-Tr-T-3'-CH₂ -O-NH-CH₂ -5'-T-3'-O-TBDPSidimer. ¹ H-NMR (CDCl₃) δ 8.73 (br s, 2, 2NH), 7.67 (s, 1 C₆ H),7.64-7.23 (m, 20, TrH, TBDPhH), 6.96 (s, 1, C₆ H), 6.23 (pseudo t, 1, T₂H_(1')), 6.11 (pseudo t, 1, T₁, H_(1')) 5.51 (br s, 1, NH), 4.16 (m, 1,T₂ H_(3')) 4.02 (m, 1, T₂ H_(4')), 3.87 (m, 1, T₁ H_(4')), 3.52 (m, 3,T1 CH_(23"), T₁ H_(5")), 3.23 (m, 1, T₁ H5'), 2.55-2.76 (m, 3, T1H_(3'), T2 H_(5') H_(5")), 2.33-2.27 (m, 1, T2 H_(2')), 2.23-2.12 (m, 2,T1 H₂ H_(3")), 1.95-1.85 (m, 1, T₂ H.sub. 2"), 1.83 (s, 3, CH₃) 1.45 (s,3, CH₃), 1.06 (s, 9, (CH₃)₃ CSi).

The latter dimer was methylated using HCHO/NaBH₃ CN/AcOH and finallydeblocked with nBu₄ NF/THF and HF/CH₃ CN in two-steps to furnish thetitle compound (65% yield). ¹ H-NMR (DMSO-d₆) δ 11.27 (br s, 2, NH);7.85 s, 1, T1 C₆ H); 7.51 (s, 1, T₂ C₆ H); 6.15 (pseudo t, 1, T₂ H₁,J_(1'-2') =7.8 Hz, J_(1'-2") 6.3 Hz); 6.00 (pseudo t, 1, T₁ H_(1'),J_(1'-2') =6.9 Hz, J_(1'-2") =4.5 Hz), 5.32 (br s, 1, OH_(3')), 5.09 (brs, 1, OH_(5')); 4.17 (m, 1, T₂ H_(3')); 3.90 (m, 1, T₂ H₄); 3.76-3.66(m, 4, T₁ H_(4'), T₁ H_(5'), CH₂ 3"); 3.60-3.52 (m, 1, T₁ H_(5")); 2.82(m, 2, T₂ H_(5'), H_(5")); 2.57 (s, 3, N-CH₃); 2.47 (m, 1, T₁ H_(3'));2.23-2.02 (m, 4, H_(2') H_(2")) 1.81 (s, 3, C₅ CH₃); 1.78 (s, 3, C₅CH₃). Anal. Calcd. for C₂₂ H₃₁ N₅ O₉.O. 5 H₂ O: C, 50.96; H, 6.22; N,13.50. Found: C, 51.01; H, 6.22; N, 13.19. MS (FAB+, glycerol) M+H⁺m/z=510.

EXAMPLE 20 Synthesis of phosphoramidate containing (3'-CH₂ -O-N(CH₃)-CH₂-5') linked oligonucleoside

3'-De(oxyphosphinico)-3'-[methyleneoxy(methylimino)]-thymidylyl-5'-O-(dimethyoxytriphenylmethyl)-(3'→5')-3'-(O-β-cyanoethyldiisopropylaminophosphiryl)thymidine

Dimethyoxytritylation of the dimer 5'-OH-T-3'-CH₂ -O-NCH₃ -CH₂-5'-T-3'-OH following the procedure described in OligonucleotideSynthesis: a practical approach, Ed. M. J. Gait, IRL Press, 1984,furnished the 5'-O-DMTr protected dimer as white foam. ¹ H-NMR (CDCl₃) δ7.67 (s, 1, H₆); 7.44-6.82 (m, 14, H₆, DMTrH); 6.20 (pseudo t, 2,H_(1')); 4.3 (m, 1, T₂ H_(3')); 4.15 (m, 1, T₂ H_(4')); 4.00 (m, 1, T₁H_(4')); 3.80 (s, 6, OCH₃); 3.77-3.23 (m, 4, T₁ H_(5') H_(5"), CH₂ 3");2.89-2.50 (m, 3, T₂ H_(5') H_(5"), T1 H₃ ⁴⁰ ); 2.62 (s, 3, N-CH₃);2.48-2.08 (m, 4, H_(2') H_(2")); 1.9 (s, 3, C₅ CH₃) 1.48 (s, 3 C₅ CH₃).

Above compound was phosphitylated following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish the title compound in 70% yield over two stepsas white powder. ¹ H NMR (CDCl₃) δ 8.25 (br s, 2, NH), 7.66 (s, 1, C₆H), 7.15-7.45 (m, 10, ArH, C₆ H), 6.8-6.9 (m, 4, ArH), 6.12 (m, 2,2C_(1') H), 3.79 (s, 6,ArOCH₃), 2.56 (s, 3, N-CH₃), 1.88, 1.44 (2s, 6,2C₅ CH₃) and other protons. ³¹ P NMR (CDCl₃) 149.42 and 148.75 ppm.

EXAMPLE 21 Synthesis of oligonucleosides having linkages that includepharmacokinetic and pharmacodynamic property modifying groups locatedtherein on

3'-De(oxyphosphinico)-3'-[methylene(benzylimino)]-thymidylyl-5'-O-(dimethyoxytriphenylmethyl)-3'→5')-3'-O-β-(cyanoethyldiisopropylaminophosphiryl)thymidine

A reductive coupling of 3'-deoxy-3'-C-formyl-5'-O-tritylthymidine (1.5mmol) with 5'-O-amino-3'-O-(t-butyldiphenylsilyl)thymidine (1.5 mmol) asdescribed in Example 9 furnished 5'-O-Tr-T-3'-CH₂ -NH-O-CH₂-5'-T-3'-O-TBDPSi dimer. This dimer was benzylated with C₆ H₅ CHO/NaBH₃CN/AcOH in the same manner as the above described methylation to yieldN-benzylated dimer 5'-O-Tr-T-3'-CH₂ -NBz-O-CH₂ -5'-T-3'-O -TBDPSi. Thelatter dimer was deblocked using nBu₄ NF/THF and HCl/MeOH methodology asdescribed in above examples to furnish deblocked dimer 5'-OH-T-3'-CH₂-NBn-)-CH₂ -5'-T-3'-OH, which on dimethoxytritylation and subsequentphosphitylation following the procedure described in OligonucleotideSynthesis: a practical approach, Ed. M. J. Gait, IRL Press, 1984, gavethe title compound (45% overall yield). ¹ H NMR (CDCl₃) δ 6.15 (pseudot, 1, T2 C_(1') H); 6.09 (m, 1, T1 C_(1') H); 3.76 (s, 6, 20CH₃); 1.7and 1.48 (2S, 6, 2-CH₃) and other protons. ³¹ p NMR (CDCl₃) 149.59 and149.23 ppm.

The phosphiltylated dimer was successfully incorporated into an oligomerusing an automated DNA synthesizer in the manner of Example 8illustrating the ability to attach of various pharmacokinetic andpharmacodynamic property modifying groups into the backbone linkageprior to the DNA synthesis of an oligonucleotide.

EXAMPLE 22 Synthesis of (3'-CH₂ -NH-CH₂ -CH₂ -5'), (3'-CH₂ -N(H₃)-CH₂-CH₂ -5'), and Phosphoramidate Derivative

3'-De(oxyphosphinico-3'-[(methyleneimino)-methylene]-5'-O-(dimethyoxytrityl)thymidylyl-3'→5')-thymidine

A reductive amination [according to the procedure of Abdel-Magid, etal., Tetrahedron Letts. 1990, 31, 5595] of3'-deoxy-3'-C-formyl-5'-O-tritylthymidine (1 mmol with1-[6'-amino-2',5',6'-trideoxy-3'-O-(t-butyldiphenylsilyl)-β-D-erythro-hexofuranosyl]thymine[1.2 mmol, prepared according to the procedure of Etzold. et al., J.C.S.Chem. Comm. 1968, 422] in presence of AcOH gave a blocked dimer5'-O-Tr-T-3'-CH₂ NH-CH₂ -CH₂ -5'-T-3'-O-TBDPSi, which on deprotection asdescribed in above examples gave 5'-OH-T-3'-CH₂ -NH-CH₂ -CH₂ -5'-T-3'-OHdimer as white powder (70% yield). ¹ H NMR (D₂ O, pH 5.6, 20° C.) δ T1thymidine unit: 7.78 (s, 1, C₆ H); 6.17 (t, 1, C₁ H); 4.45 (m, 1, C_(3')H); 4.08 (m, 1 , C_(4'H)); 4.00, 3.72 (m, 2, C_(5'), 5" H); 2.9 (m, 2C_(6'),6" H); 2.34 (m, 2, C_(2'),2 H); 1.77 (s, 3, CH₃); T2 thymidineunit: 7.47 (s, 1 C₆ H); 6.07 (t, 1, C_(1') H); 3.89 (m, 2, C_(5') 5" H);3.79 (m, 1, C_(4') H); 2.89 (m, 1, C_(3") H); 2.38 (m, 1, C_(2') H);2.32 (m, 1, C_(3') H); 1.72 (s, 3, CH₃); and 2.68 (s, N-CH₃).

Pka determination:

The sensitivity of the proton chemical shift of the N-Me group of theforegoing dimer to change in response to change in pH was measured byNMR as an indicator of the pka of the backbone amine. The chemical shiftmoved downfield as the amino group was protonated. A 4 mg sample of5'-OH-T-3'-CH₂ -NCH₃ -CH₂ -CH₂ -5'-T-3'-OH dimer was dissolved in 0.6 mlof 30 mM bicarbonate buffer. The pH was varied between 5.1 and 10.0using 0.1N NaOH in 6-steps. The chemical shift of the N-methyl protonvaried between 2.26 and 2.93 ppm, giving rise to a pka of 7.8±0.1. Whilewe do not wish to be bound by theory, it is thus believed that atphysiological pH this backbone will be protonated.

3'-De(oxyphosphinico-3'-[methylene(methylimino)-methylene]-5'-O-(dimethyoxytrityl)-thymidylyl-(3'→5')-3'-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The proceeding dimer was methylated using HCHO/NaBH₃ CN in AcOH tofurnish 5'-OH-T-3'-CH₂ -N(CH₃)-CH₂ -CH₂ -5'-T-3'-OH dimer, which ondimethoxytritylation and phosphitylation following the proceduredescribed in Oligonucleotide synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, gave the title compound as foam (68% yield). ¹ HNMR (CDCl₃) δ 6.12 (m, 2, 2C_(1') H); 2.15, 2.14 (2s, 3, N-CH₃); 1.88,1.45 (2s, 6, 2 C₅ CH₃) and other protons. ³¹ P NMR (CDCl₃) 149.49 and148.96 ppm.

EXAMPLE 23 A (3'-CH₂ -N(labile blocking group)-O-CH₂ -5') dimer andphosphoramidate derivative - a dimer Incorporating a3'-de(oxyphosphinico)-3'-(methyleneimino) (3→5') linkage having a labileN-protecting group for regeneration of a (3'-CH₂ -NH-O-CH₂ -5) linkage

3'-De(oxyphosphinico)-3'-[methylene(phenoxyacetylimino)]-thymidylyl-(3'.fwdarw.5')-thymidine

to a stirred solution of 5'-O-Tr-T-3'-CH₂ -NH-O-CH₂ -5'-T-3'-O-TBDPSi (1mmol, prepared according to the procedure of Depart, et al., TetrahedronLetts. 1992, 33, 2645) in dry pyridine (10 ml) was addedphenoxyacetylchloride (1.2 mmol). After 12 h, the products were dilutedwith CH₂ Cl₂ (200 ml) and washed with sat. NaHCO₃ (3×50 ml), water (2×50ml) and dried (MgSO₄). The CH₂ Cl₂ extract was concentrated and residuepurified by silica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(9:1, v/v), pooling of appropriate fractions and evaporation furnished5'-O-Tr-T-3'-CH₂ -N(COCH₂ OPh)-O-CH₂ -5'-T-3'-O-TBDPSi dimer as whitefoam. ¹ H NMR (DMSO-d₆) δ 11.35 (br s, 2, NH); 7.6-6.65 (m, 32Tr, TBDPS,phenoxyacetyl, C₆ H); 6.3 (pseudo t, 1, H_(1') ); 6.03 (pseudo t, 1,H_(1')); 4.5 (m, 2, CH₂); 4.3 (m, 1, T₂ H₃); 3.9-3.3 (m, 6, T₁ H_(4'),T₂ H_(4'), T₂ H_(4'), T₂ H_(5') H_(5"), CH₂ 3"); 3.10 (m, 2T, H_(5')H_(5")); 2.65 (m, 1, T₂ H_(3')); 2.2-2.05 (m, 4, H_(2') H_(2")); 1.58(s, 3, CH₃); 1.4 (s, 3, CH₃); 1.02 (s, 9, (CH₃)₃ CSi).

The foregoing dimer was sequentially deblocked with HF (48%)/CH₃ CN(5:95, v/v) treatment to remove the trityl group, and the product ontreatment with nBu₄ NF/THF removed the silyl group to furnish titlecompound as white powder (70% yield for 3-steps). ¹ H NMR (DMSO-d₆) δ11.35 (br s, 1, NH); 11.25 (br s, 1, NH) 7.92 (s, 1, C₆ H); 7.5 (s, 1,C₆ H); 7.2-6.8 (m, 5, ArH); 6.23 (pseudo t, 1, H_(1')); 5.98 (dd, 1,H_(1')); 5.45 (d, 1, OH_(3')), 5.15 (t, 1, OH_(5')); 4.9 (m, 2, CH₂);4.3-3.5 (m, 9, T₂ H_(3'), H_(4'), H_(5') H_(5"), CH_(23")); 2.6 (m, 1,T₁ H_(3')); 2.25-2.00 (m, 4, H_(2') H_(2")) ; 1.75 (s, 3, CH₃); 1.65 (s,3, CH₂).

The latter dimer was dimethoxytritylated as per the procedure ofdescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, 1984IRL Press, to furnish 5'-O-DMT-T-3'-CH₂ -N-(COCH₂ OPh)-O-CH₂-5'-T-3'-OH as pale yellow colored foam. ¹ H NMR (DMSO d₆) δ 11.3 (br s,2, NH); 7.55 (s, 1, C₆ H). 7.45 (s, 1C₆ H); 7.38-6.75 (m, 18, DMTrH,phenoxyacetyl-H); 6.22 (pseudo t, 1, T₂ H_(1')); 6.05 (pseudo t, 1,T₁H_(1')); 4.75-4.60 (m, 2, CH₂); 4.25 (m, 1, T₂ H_(5')); 4.18 (m, 1, T₂H_(3')); 4.05 (m, 1, T₂ H_(5")); 3.9 (m, 2, H_(4')); 3.8-3.6 (m, 2, CH₂3"); 3.65 (s, 6, 2OCH₃) 3.2 (m, 2, T₁, H_(5') H_(5")) 2.82 (m, 1, T₁H_(3')); 2.3-2.05 (m, 4, H_(2') H_(2")); 1.6 (s, 3, T₂ CHCH₃); 1.38 (s,3, T1 CH₃).

The above dimer on phosphitylation following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, 1984,IRL Press, furnished the phosphoramidate derivatized dimer (appropriatefor use on DNA synthesizer) as a foam (75% in 2 steps). ¹ H NMR (CDCl₃)δ 7.62 (s, 1, C₆ H); 7.2-7.45 (2m, 12, ArH); 6.77-7.05 (3m, 7, ArH, C₆H); 6.15 (pseudo t, 1, C_(1') H); 6.05 (t, 1, C_(1'H)); 4.7 (m, 2,2C_(4') H); 3.74 (2s, 6, 2ArOCH₃); 2.95 (m, 1, C_(3") H); 1.78, 1.77(2s, 3, C₅ CH₃); 1.41 (s, 3, C₅ CH₃, and other protons, ³¹ P NMR (CDCl₃)1.49.76 and 149.56 ppm.

EXAMPLE 24 Regeneration of (3'-CH₂ -NH-O-CH₂ -5') linkage from (3'-CH₂-N(labile blocking group)-CH₂ CH₂ -5') linkage In an oligonucleotide

The phosphitylated dimer of Example 23 will be incorporated within anoligonucleotide as per the procedure of Example 8. After completion ofthe oligonucleotide on the support, the oligonucleotide is cleaved fromthe support utilizing standard ammonium hydroxide conditions. Concurrentwith the cleavage from the support the ammonium hydroxide treatment willfurther cleave the phenoxyacetyl blocking group from the imino nitrogenof the incorporated (3'-CH₂ -N(COCH₂ OPh)-O-CH₂ -5') oligonucleosidedimer to yield the (3'-CH₂ -NH-O-CH₂ -5') linked oligonucleoside dimerwithin the oligonucleotide structure.

EXAMPLE 25 5'-O-(t-Butyldimethylsilyl)-3'-O-Phthalimidothymidine, 2

To a solution of 5'-O-t-butyldimethylsilylthymidine [1, 21.36 g, 60mmol, prepared according to the procedure of Nair, et al., Org. Prep.Procedures Int. 1990, 22, 57 in dry THF (750 ml)], triphenylphosphine(17.28 g, 66 mmol) and N-hydroxyphthalimide (10.74 g, 66 mmol) wereadded. The solution was cooled to 0° C. and diisopropylazodicarboxylate(15.15 g, 75 mmol) was added dropwise over a period of 3 hr whilestirring under nitrogen. The reaction mixture was then stirred at roomtemperature for 12 hr. The solution was evaporated and the residue wasdissolved in CH₂ Cl₂ (750 ml), extracted with sat. NaHCO₃ (200 ml), andwater (200 ml), dried (MgSO₄), filtered and concentrated to furnishyellow oily residue. Silica gel column chromatography (100% hexanes, andthen hexanes:Et₂ O gradient to 90% Et₂ O) of the residue gave compound 2as a colorless glass (18.68 g, 62%); ¹ H NMR (CDCl₃) δ 0.05 [2s, 6,(CH₃)₂ ], 0.91 [s, 9, (CH₃)₃ ], 2.0 (s, 3, CH₃), 2.5-2.65 (m, 2, 2'CH₂),4.05-4.2 (m, 2, 5'CH₂), 4.25-4.35 (m, 1, 4'H), 5.0 (m, 1, 3'H), 6.15 (m,1, 1'H), 8.6 (br s, 1, NH), and aromatic protons, Anal. Calcd, for C₂₄H₃₁ N₃ O₇ Si: C, 57.46H, 6.23; N, 8.37. found : C, 57.20; H, 6.26; N,8.27.

EXAMPLE 26 3'-O-Amino-5'-O-(t-Butyldimethylsilyl)thymidine, 3

Cold methylhydrazine (1.6 ml, 30 mmol) was added to a stirred solutionof 5'-O-(t-butyldimethylsilyl)-3'-O-phthalimidothymidine (2, 4.6 g, 9.18mmol) in dry CH₂ Cl₂ (60 ml) at 5°-10° C. After 10 minutes whiteprecipitation of 1,2-dihydro-4-hydroxy-2-methyl-1-oxophthalizineoccurred. The suspension was stirred at room temperature for 1 h. Thesuspension was filtered and precipitate washed with CH₂ Cl₂ (2×20 ml).The combined filtrates were concentrated and the residue purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(100:0→97:3, v/v) furnished the title compound (3.40 g, 100%) as whitesolid. Crystallization from CH₂ Cl₂ gave white needles, m.p. 171° C.; ¹H NMR (CDCl₃) δ 0.05 [s, 6, (CH₃)₂ ], 0.90 [s, 9, (CH₃)₃, 2.22-2.58 (2m,2, 2'CH₂), 3.9-4.08 (m, 3, 5'CH₂, and 3'H) 4.30 (m, 1, 4'H) 5.5 (br s,2, NH₂) 6.2 (m, 1, 1'H) 7.45 (s, 1, C₆ H) 8.9 (br s, 1, NH). Anal.Calcd. for C₁₆ H₂₉ N₃ O₅ Si: C, 51.72; H, 7.87; N, 11.32. found: C,51.87, H, 7.81; N, 11.32.

EXAMPLE 27 3'-O-Aminothymidine, 4

3'-O-Amino-(t-butyldimethylsilyl)thymidine was deblocked with (Bu)₄NF/THF in standard way to furnish compound 4 (72%). Crystallized fromether/hexanes/ethanol as fine needles, mp 81° C. ¹ h NMR (Me₂ SO-d₆) δ1.78 (s, 3, CH₃), 2.17 and 2.45 (2m, 2, 2'CH₂), 3.70 (m, 2, 5'CH₂), 3.88(m, 1, 4'H), 4.16 (m, 1, 3'H), 4.8 (br s, 1, 5'OH), 6.05 (dd, 1, 1'H),6.2 (br s, 2 NH₂), 7.48 (s, 1, C₆ H), and 11.24 (br s, 1NH). Anal.Calcd. for C₁₀ H₁₅ N₃ O₅ : C, 46.69; H, 5.87; N, 16.33; found: C, 46.55;H, 5.91; N, 16.21.

EXAMPLE 283'-O-Dephosphinico-3'-O-(Methylimino)thymidylyl-(3'→5')-5'-Deoxythymidine,9 Step 1.

3'-O-Amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 1.85 g, 5 mmol),3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde [5, 2.39 g, 5 mmol;freshly prepared by following the method of Camarasa, et al.,Nucleosides and Nucleotides 1990, 9, 533] and AcOH (0.25 ml) werestirred together in CH₂ Cl₂ (50 ml) solution at room temperature for 2h. The products were then concentrated under reduced pressure to givethe intermediate oxime linked dimer, compound 6.

Step 2.

The residue obtained from Step 1 was dissolved in AcOH (25 ml). NaCNBH₃(1.55 g, 25 mmol, in 3-portions) was added to the stirred AcOH solutionat room temperature. The solution was stirred for 30 min to give theintermediate imine linked dimer, compound 7.

Step 3.

Aqueous HCHO (20% , 2 ml, 66 mmol) and additional NaCNBH₃ (1.55 g, 25mmol, in 3portions) was added to the stirred reaction mixture of Step 2at room temperature. After 2h, the solution was diluted with EtOH (100ml), and resulting suspension was evaporated under reduced pressure. Theresidue was dissolved in CH₂ Cl₂ (150 ml) and then washed successivelywith 0.1M HCl (100 ml), saturated aqueous NaHCO₃ (100 ml), and water(3×50 ml). The dried (MgSO₄) CH₂ Cl₂ solution was evaporated to giverude methylated imine linked dimer 8.

Step 4.

The residue from Step 3 was dissolved in the THF (30 ml) and a solutionof (Bu)₄ NF (1M in THF, 10 ml) was added while stirring at roomtemperature. After 1 h, the reaction mixture was evaporated underreduced pressure and the residue was purified by short columnchromatography. The appropriate fractions, which eluted with CH₂ Cl₂:MeOH (8:2, v/v) were pooled and evaporated to give compound 9 as a foam(0.74 g, 30%). ¹ H NMR (Me₂ SO-d₆) δ 1.78 (s, 6, 2CH₃), 2.10 (m, 4,2'CH₂), 2.5 (s, 3, N-CH₃), 2.8 (m, 2, 5'-N-CH₂), 3.6-4.08 (5m, 6, 5'CH₂, 4' CH, 3' CH), 4.75 and 5.3 (2 br s, 2, 3' and 5' OH), 6.02 (d, 1,1'H), 6.1 (t, 1, 1'H), 7.4 and 7.45 (2s, 2, 2C₆ H), 11.3 (br s, 2, NH).

EXAMPLE 29 Methyl3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-5-C-Formyl-α/β-erythro-Pentofuranoside,23

2-Deoxy-D-ribose, 21, was modified to methyl2-deoxy-a/βD-erythro-pentofuranoside (prepared according to the methodof Motawai, et al., Liebigs Ann. Chem. 1990, 599-602), which onselective tosylation followed by 3-O-silylation gave methyl3-O-(t-butyldimethylsilyl)-2-deoxy-5-O-tosyl-α/β-D-erythro-pentofuranosidein overall 70% yield. The latter compound on iodination followed bycyanation gave the corresponding 5-C-cyano intermediate compound 22, asa syrup. ¹ H NMR (CDCl₃) δ 1.05 (s, 9, (CH₃)₃), 1.9-2.38 (m, 4, 2 CH₂),3.3 and 3.4 (2s, 3, OCH₃), 3.98-4.30 (3m, 2, 3, 4-CH), 4.95 and 5.05(2m, 1, 1H), 7.4 and 7.7 (2m, 10, Ph H). IR (neat) 2253 cm⁻¹ (CH₂ CN)].Compound 22 (stored at 0° C. without any degradation) was reduced(DIBAL-H) freshly every time as and when the title compound 23 wasrequired.

EXAMPLE 305'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'[(Methyleneamino)oxy]adenosine,27;5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-[(Methyleneamino)oxy]cytidine,28; and5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-[(Methyleneamino)oxy]guanosine,29

3'-O-Amino-2'-deoxyadenosine, compound 24, 3'-O-amino-2'-deoxycytidine,compound 25, and 3'-O-amino-2'-deoxyguanosine, compound 26, prepared asper the procedures of European Patent Application 0 381 335 or in amanner analogous to the preparation of compound 4 by the procedure ofExample 27 above, are blocked at their 5' position with at-butyldimethylsilyl group according to the procedure of Nair, et al.,Org. Prep. Procedures Int. 1990, 22, 57, to give the corresponding3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyadenosine,3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxycytidine and3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyguanosine nucleosideintermediates. Treatment of the blocked intermediate as per theprocedures of Example 5 or as per the procedure of Preparation example28 of European Patent Application 0 381 335 gives the corresponding5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]adenosinecompound 27;5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]cytidine,compound 28; and5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]guanosine,compound 29.

EXAMPLE 31

3'-O-(t-Butyldiphenylsilyl)thymidine-6'-Aldehyde, 31

The title compound is prepared by homologation of the above described3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde (compound 5) utilizingthe procedure of Barton, et al., Tetrahedron Letters 1989, 30, 4969. The5'-aldehyde, compound 5, is treated via a Witig reaction with(methoxymethylidene)triphenylphosphate. The resulting enol ether,compound 30, is hydrolyzed with Hg(OAc)₂, KI, H₂ O and THF according tothe procedure of Nicolaou, et al., J. Am. Chem. Soc. 1980, 102, 1401 tofurnish the compound 31.

EXAMPLE 32

5'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Nitrilomethylidyne)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl-5'-Deoxythymidine,32

The title compound is prepared by reaction of compound of 31 andcompound 3 in the manner of Example 28, Step 1 to furnish the dimericoligonucleoside having an oxime backbone.

EXAMPLE 33

3'-O-Dephosphinico-3'-O-[(Methylamino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method B

Compound 32 when treated as per the procedure of Steps 2 and 3 ofExample 28 will also yield compound 14.

EXAMPLE 34

Methyl3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-thymidylyl-(3'→5')-3-O-(T-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,33

Compound 23 and compound 3 are linked utilizing the procedure of Example28, Steps 1 to couple the sugar and the nucleoside via an oxime linkage.The resulting oxime linkage is then reduced utilizing the procedure ofExample 28, Step 2 to an iminomethylene linkage and this linkage, inturn, when N-alkylated via the procedure of Example 28, Step 3 willyield compound 33.

EXAMPLE 35

Acetyl5'-O-Benzoyl-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]thymidylyl-(3'→5)-3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,34

Compound 33 will be treated with benzoyl chloride according to theprocedure of Jenkins, et al., Synthetic Procedures in Nucleic AcidChemistry, Zorbach and Tipson, Ed., Vol. 1, John Wiley & Sons, Pg. 149,to benzoylate the free 5'-hydroxyl of compound 33 which is hydrolyzedand acylated in situ according to the procedure of Baud, et. al,Tetrahedron Letters 1990, 31, 4437 to yield compound 34.

EXAMPLE 36

5'-Benzoyl-3'-O-Dephosphinico-3'O-[Methylimino)methylene]thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,35

Compound 34 is reacted with silylated thymine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene to yield5'-O-benzoyl-3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine,compound 35 as an anomeric mixture.

EXAMPLE 37

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,15

Method C

Compound 35 when treated with methanolic ammonia will also yieldcompound 14. Further treatment as per the procedure of Example 9 willyield the fully deblocked dimer, from which anomerically pure compound15 will be isolated by chromatography.

EXAMPLE 38

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyadenosine,36

Compound 34 is reacted with silylated adenine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyadenosine,36.

EXAMPLE 39

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxycytidine37

Compound 34 is reacted with silylated cytosine as per the procedure ofBaud, et al., Tetrahedron Letters, 1990 31, 4437, utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxycytidine,37.

EXAMPLE 40

3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'O-(t-Butyldiphenylsilyl)-5'-Deoxyguanosine38

Compound 34 is reacted with silylated guanine as per the procedure ofBaud, et al., Tetrahedron Letters 1990, 31, 4437 utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl-5'-deoxyguanosine,38.

EXAMPLE 41

A-(3'→5')-T; A-(3'→5')-A; A-(3'→5')-C; and A-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoadenosine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is adenine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the A-T, A-A, A-C and A-G dimers,respectively, of a structure equivalent to that of compound 13 where Bxiis adenine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 42

C-(3'→5')-T; C-(3'→5')-A; C-(3'→5')-C; and C-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminocytidine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is cytidine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the C-T, C-A, C-C and C-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis cytosine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 43

G-(3'→5')-T; G-(3'→5')-A; G-(3'→5')-C; and G-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoguanosine intermediate of Example 30will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is guanine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the G-T, G-A, G-C and G-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis guanine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 44

Trimeric, Tetrameric, Pentameric, Hexameric And Other Higher OrderOligonucleosides Having a Selected Nucleoside Sequence

The dimers of Examples 21, 23, 24, 25, 26, 27 and 28 are extended byreaction with the5'-(t-butyldimethylsilyl)-3'-deoxy-3'-[(methyleneamino)oxy] nucleosides,compounds 10, 27, 28 and 29, of Examples 5 and 15 to form trimersutilizing the looping sequence of reactions of Examples 10, 11 and 12.Iteration of this reaction sequence loop adds a further nucleoside tothe growing oligonucleoside per each iteration of the reaction sequenceloop. The reaction sequence loop of Examples 10, 11 and 12 is repeated"n" number of times to extend the oligonucleoside to the desired "n+1"length. The final 3'-blocked oligonucleoside when treated as per theprocedure of Example 9 to remove the terminal3'-O-(t-butyldiphenylsilyl) blocking group will yield the fullydeblocked oligonucleoside of the selected nucleoside sequence andlength.

EXAMPLE 45

6'-Amino-6'-Deoxy-5'-Homothymidine, 42;6'-Amino-4',6'-Dideoxy-5'-Homoadenosine, 43;6'-Amino-2',6'-Dideoxy-5'-Homocytidine, 44; and6'-Amino-2',6'-Dideoxy-5'-Homoguanosine, 45 (Via An Intramolecular FreeRadical Reaction)

Deblocking of compound 10 is effected by treatment with Bu₄ NF in THF.The resulting compounds 39 (also reported in Preparation example 4 ofEuropean Patent application 0 381 335 A1) will be iodinated upontreatment with methyltriphenoxyphosphonium iodide as per the procedureof Verheyden, et al., J. Org. Chem. 1970, 35, 2119 to furnish5'-deoxy-5'-iodo-3'-O-methyleneaminothymidine, compound 40. Compound 40when subjected to an intramolecular free radical reaction according tothe procedure of Curran, D. P., Radical Addition Reactions, InComprehensive Organic Synthesis: Trost, B. M. and Fleming, I., Eds.,vol. 4, p 715-832, Pergamon Press, Oxford (1991), will give thecorresponding 3'-O-isoxazolidinethymidine, compound 41 which on DIBAL-Hreduction will yield 6'-amino-5'-homothymidine, compound 42 [the3'-(t-butyldimethylsilyl) derivative of this compound is reported inRawson, et al., Nucleosides & Nucleotides 1990, 9, 89].

When reacted in a like manner compounds 27, 28 and 29 will give6'-amino-5'-homoadenosine, compound 43; 6'-amino-5'-homocytidine,compound 44; and 6'-amino-5'-homoguanosine, compound 45.

EXAMPLE 46

3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-C-Allylthymidine 46

A stirred solution of3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iododthymidine (12, 1.77 g, 3mmol), allytributyltin (2.97 g, 9 mmol) and AIBN (0.54 g, 3.3 mmol) indry toluene (30 ml) was degassed completely and heated at 65° C. for 6hr. The solution was cooled and concentrated under vacuo. The residuewas purified by silica gel column chromatography and on elution withhexanes:EtOAc (1:1, v/v) furnished the title compound as homogeneousmaterial. Appropriate fractions were pooled and evaporated to furnish46, 0.75 g of a white foam, 50% yield. The structure was confirmed by ¹H NMR.

EXAMPLE 47

3'-O-(t-Butyldiphenylsilyl)-5-Deoxy-7'-C-Aldehydothymidine 47

A solution of 46 (1 mmol), OsO₄ (0.1 mmol) and n-methylmorpholine oxide(2 mmol) in diethyl ether (4 ml) and water (2 ml) are stirred for 18 hrat room temperature. A solution of NaIO₄ (3 ml) is added and thesolution further stirred for 12 hr. The aqueous layer is extracted withdiethyl ether. Evaporation of the organic layer will give the crudealdehyde 47.

EXAMPLE 48

N3-Benzoyl-1-(5'-O-Dimethoxytrityl-3'-O-Trifluoromethylsulfonyl-threo-Pentofuranosyl)thymine,50

The method of Horwitz, et al., J. Org. Chem. 1964, 29, 2076 with beutilized to prepare the title compound withthreo-3'-O-trifluoromethanesulfonate. Also, reaction conditions ofFleet, et al., Tetrahedron 1988, 44, 625, will furnish a 3'-leavinggroup in the threo configuration.

EXAMPLE 49

6'-O-Phthalimido-5'-Homothymidine, 52

To a stirred mixture of 5'-homothymidine [Etzold, et al., ChemicalCommunications 1968, 422] (51, 1.28. g, 5 mmol), N-hydroxyphthalimide(1.09 g, 6.6 mmol) and triphenylphosphine (1.75 g, 6.6 mmol) in dry DMF(25 ml) will be added diisopropylazodicarboxylate (1.5 ml, 7.5 mmol)over a period of 30 min at 0° C. The stirring is continued for 12 hr atroom temperature. The solvent is evaporated under vacuo and the residueis washed with diethyl ether (2×50 ml). The residue will then besuspended in hot EtOH (50 ml), cooled and filtered to give the titlecompound 52.

EXAMPLE 50

6'-O-Phthalimido-3'-O-(t-Butyldiphenylsilyl)-Homothymidine 53

Compound 52 will be treated with t-butyldiphenylchlorosilane in pyridineand imidazole in a standard manner to afford the title compound 53.

EXAMPLE 51

6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine, 54

To a stirred solution of compound 53 in dry CH₂ Cl₂ is addedmethylhydrazine (3 mmol) under anhydrous conditions at room temperature.The solution is stirred for 12 hr, cooled (0° C.) and filtered. Theprecipitate will be washed with CH₂ Cl₂ and the combined filtrates willbe concentrated. The residue is purified by flash column chromatography(silica gel, 20 g). Elution with CH₂ Cl₂ :MeOH, 9:1, v/v) with furnishthe title compound 54.

EXAMPLE 52

3'-De(oxophosphinico)-3'-(iminoxymethylene)-5'-Tritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-deoxythymidine,55

6'-O-Amino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine, 54, isconverted to the corresponding urethane with ethyl chloroformate (CH₂Cl₂ -saturated NaHCO₃) utilizing the stereospecific conditions of Yan,et al., J. Am. Chem. Soc. 1991, 113, 4715. The residue of this reactionwill then be stirred in CH₂ Cl₂ with compound 50. The products are thenconcentrated in vacuo to yield the dimeric oligonucleoside, compound 55.

EXAMPLE 53

3'-De(oxophosphinico)-3'-[Methyl(iminoxymethylene)]-5'-Tritylthymidylyl-(3'→5')-3'O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,56

Compound 55 will be N-alkylated as per the conditions of Step 3 ofExample 4 to yield the N-alkylate iminooxymethylene linked dimericoligonucleoside 56.

EXAMPLE 54

3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-dimethoxytritylthymidylyl-(3'→5')-5'-Deoxythymidine,57

The 5'-O-Trityl and the 3'-O-(t-butyldiphenylsilyl) protecting groups ofcompound 56 will be removed by treatment with trifluoroacetic acid andthe residue dimethoxytritylated as per the procedure of Sproat, B. S.and Lamond, A. I., 2'-O-Methyloligoribonucleotides: Synthesis andApplications, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, pg. 55 (1991), to give the title compound.

EXAMPLE 55

3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(diisopropyl)phosphiryl]-5'-Deoxythymidine,58

Compound 57 (1.89 mmol) will be dissolved in anhydrous dichloromethaneunder an argon atmosphere. Diisopropylethylamine (0.82 ml, 4.66 mmol) isadded and the reaction mixture cooled to ice temperature.Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 ml), 4.03 mmol) isadded to the reaction mixture and the reaction mixture is allowed towarm to 20° C. and stirred for 3 hr. Ethylacetate (80 ml) andtriethylamine (1 ml) are added and the solution is washed with brinesolution three times (3×25 ml). The organic phase is separated and driedover magnesium sulfate. After filtration of the solids the solvent isevaporated in vacuo at 20° C. to an oil that will then be purified bycolumn chromatography using silica and a solvent such as hexane-ethylacetate-triethylamine (50:40:1) as eluent. The fractions are thenevaporated in vacuo and the residue will be further evaporated withanhydrous pyridine (20 ml) in vacuo (1 torr) at 26° C. in the presenceof sodium hydroxide for 24 hr to yield the title compound 58.

EXAMPLE 56

5'-Amino-5'-Homothymidine, 60

5'-Amino-3'-O-(t-butyldimethylsilyl)-5'-homothymidine 59 is prepared asper Rawson, et al., Nucleosides & Nucleotides 1990, 9, 89. Thet-butyldimethylsilyl group will be removed as per the procedure of Step4 of Example 4 to give the title compound.

EXAMPLE 57

5'-Methylamino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine, 62

Compound 60 is t-butyldiphenylsilated as per the procedure of 37 to give5'-Amino-3'-O-(t-butyldiphenylsilyl)- 5'-homothymidine, compound 61,which will then be treated as per the procedure of Step 3 of Example 4alkylate the 5'-amino group to yield the title compound 62.

EXAMPLE 58

3'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,64

5'-Methylamino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine 62 (1 mmol)will be added to aqueous sodium hypochloride (4 mmol) to furnish achloramide intermediate. The chloramide intermediate is cooled (0° C.)and treated with 5'-O-monomethoxytrity-3'-thiothymidine (0.9 mmol),compound 63, prepared as per Cosstick, et al., Nucleic Acids Res. 1990,18, 829. The reaction mixture is worked up utilizing the procedure ofBarton, et al., J. Org. Chem. 1991, 56, 6702 and the residue will bepurified by chromatography to give the title compound 64.

EXAMPLE 59

3'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-5'Deoxythymidine,65

Compound 64 will be deblocked at the terminal 3' position utilizing theas per the procedure of Step 4 of Example 4 to give compound 65.

EXAMPLE 60

3'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'[(β-Cyanoethoxy)-N-(diisopropyl)phosphortityl]-5'-Deoxythymidine66

Compound 65 will be phosphitylated as per the procedure of Example 55 togive the title compound 66.

EXAMPLE 61

5'-O-(t-Butyldimethylsilyl)-3'-De(oxyphosphinico)-3'-(Imino-1,2-Ethanediyl)thymidylyl-(3'→5')3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,68

3'-Amino'-5'-O-(t-butyldimethylsilyl)-3'-deoxythymidine, compound 67,prepared according to Matsuda, et al., Nucleoside & Nucleotides 1990, 9,587 will be reductively coupled with compound 47 in the presence of acatalytic amount of acid as per the procedure of Magid, et. al,Tettrahedron Letters, 1990, 31, 5595, to yield the Schiff's baseintermediate that is reduced in situ to give the amino linkage of thetitle compound 68.

EXAMPLE 62

3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,69

Compound 68 will be methylated and deblocked at the 5' position as perthe procedure of Step 3 of Example 4 to yield the N-alkylated5'-deblocked dimer, compound 69.

EXAMPLE 63

3'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,70

Compound 69 will be dimethoxytritylated as per the procedure of Sproat,B. S. and Lamond, A. I., 2'-O-Methyloligoribonucleotides: Synthesis andApplications, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, 1991, pg. 55.

EXAMPLE 64

3'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-5'-Deoxythymidine,71

The dimethoxytritylated intermediate, compound 70 when deblocked at the3' terminus as per the procedure of Step 4 of Example 4 will givecompound 71.

EXAMPLE 65

3'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(diisopropyl)phosphiryl]-5'-Deoxythymidine,72

Compound 71 will be phosphitylated as per the procedure of Example 55 togive the title compound 72.

EXAMPLE 66

2'-O-Methylhomoadenosine, 74

Homoadenosine, 73, prepared as per the procedure of Kappler, F. andHampton, A., Nucleic Acid Chemistry, Part 4, Ed. L. B. Townsend and R.S. Tipson, Wiley-Interscience Publication, 1991, pg. 240, will beblocked across its 3' and 5' hydroxyl groups with a TIPS, i.e.,tetraisopropylsilyl, blocking group followed by alkylation as per theprocedures described in U.S. patent application Ser. Nos. 566,977, filedAug. 13, 1990 and PCT/US91/05720, filed Aug. 12, 1991. Removal of theTIPS group with tetra-n-butylammonium fluoride as per the procedure ofStep 4 of Example 4 will yield the title compound 74.

EXAMPLE 67

6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homoadenosine, 75

Compound 74 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 75.

EXAMPLE 68

3'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-2'-O-Methyladenosine,76

Compound 75 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 76.

EXAMPLE 69

3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)-]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl]-5'-Deoxy-2'-O-Methyladenosine,77

Compound 76 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 77.

EXAMPLE 70

6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-Deoxy-5'-Homoaristeromycin, 79

(-)-2'-Deoxy-5'-homoaristeromycin, compound 78, (the carbocyclic analogof 5'-homo-2'-deoxyadenosine) is prepared as per the procedure of Jones,et al., J. Chem. Soc. Perkin Trans. 1988, 1, 2927. Compound 78 will betreated as per the procedure of Examples 36, 37 and 38 to yield the6'-O-amino-3'-blocked carbocyclic analog of 5'-homo-2'-deoxyadenosine,compound 79.

EXAMPLE 71

3'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'O-(t-Butyldiphenylsilyl)-2',5'-Dideoxyaristeromycin,80

Compound 79 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 80.

EXAMPLE 72

3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Dyanoethoxy)-N-(Diisopropyl)phosphiryl]-2',5'-Dideoxyaristeromycin,81

Compound 80 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 81.

EXAMPLE 73

6'-O-Amino-2'-O-Butyl-5'-Homoaristeromycin, 82

(-)-5'-Homoaristeromycin, compound 78, will be blocked with a TIPSgroup, alkylated and deblocked as per the procedure of Example 70 toyield compound 82.

EXAMPLE 74

6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Homoaristeromycin,83

Compound 82 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 83.

EXAMPLE 75

3'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Deoxyaristeromycin,84

Compound 83 will be treated and reacted with compound 50 as per theprocedure of Example 65 to yield the title compound 84.

EXAMPLE 76

3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl]-2'-O-Butyl-5'-Deoxyaristeromycin,85

Compound 84 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 85.

EXAMPLE 77

(+)-1-[(1R,3S,4S)-3-Azido-5-Dimethoxytrityl-4-(Hydroxymethyl)-Cyclopentyl]-5-Methyl-2,4-(1H,3H)-Pyrimidindione,87

(+)-1-[1R,3S,4S)-3-Azido-4-(hydroxymethyl)-cyclopentyl]-5-methyl-2,4-(1H,3H)-pyrimidindione,compound 86, prepared as per the procedure of Bodenteich, et al.,Tetrahedron Letters 1987, 28, 5311, will be dimethoxytritylatedutilizing dimethoxytrityl chloride in pyridine at room temperature togive the title compound 87.

EXAMPLE 78

(+)-1-[(1R,3S,4)-3-Amino-4-(Dimethoxytrityloxymethyl)-Cyclopentyl]-5-Methyl-2,4-(1H,3H)-Pyrimidindione,88

Compound 87 will be reduced with Ph₃ P in pyridine at room temperatureas per the procedure of Hronowski, et al., J. Chem. Soc., Chem. Commun.1990, 1547, to give the carbocyclic analog of3'-amino-5'-dimethoxytrityl thymidine, compound 88.

EXAMPLE 79

1-{(1R,3S,4S)-3-[Imino-2-(5'-Deoxythymidylyl-5'-yl)-1,2-Ethanediyl]-4-(Dimethoxtrityloxymethyl)-Cyclopentyl}-5-Methyl-2,4-(1H,3H)-Pyrimidindione,89

Compound 88 will be reacted with compound 47 as per the procedure ofExample 74 to yield the title compound 89.

EXAMPLE 80 Synthesis of Oligonucleotides Using A DNA Synthesizer

Solid support oligonucleotide and "oligonucleotide like" syntheses areperformed on an Applied Biosystems 380 B or 394 DNA synthesizerfollowing standard phosphoramidite protocols and cycles using reagentssupplied by the manufacture. The oligonucleotides are normallysynthesized in either a 10 μmol scale or a 3×1 μmol scale in the"Trityl-On" mode. Standard deprotection conditions (30% NH₄ OH, 55° C.,16 hr) are employed. HPLC is performed on a Waters 600E instrumentequipped with a model 991 detector. For analytical chromatography, thefollowing reverse phase HPLC conditions are employed: Hamilton PRP-1column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0; solvent B: 45 mm TEAAwith 80% CH₃ CN; flow rate: 1.5 ml/min; gradient: 5% B for the first 5minutes, linear (1%) increase in B every minute thereafter. Forpreparative purposes, the following reverse phase HPLC conditions areemployed: Waters Delta Pak Waters Delta-Pak C₄ 15 μm, 300 A, 25×100 mmcolumn flow rate: 5 ml/min; gradient: 5% B for the first 10 minute,linear 1% increase for every minute thereafter. Following HPLCpurification, Oligonucleotides are detritylated and further purified bysize exclusion using a Sephadex G-25 column.

EXAMPLE 81 HIGHER ORDER MIXED OLIGONUCLEOSIDES-OLIGONUCLEOSIDES ANDMIXED OLIGONUCLEOSIDES-OLIGONUCLEOTIDES

A. Solution Phase Synthesis Of3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-3'-Phosphorothioate-Thymidylyl-(3'→5')-3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-5'-Deoxythymidine,90, A Mixed Oligonucleoside-Oligonucleotide-Oligonucleoside PolymerIncorporating A Nucleotide Linkage Flanked At Its 5' Terminus By A3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)] LinkedOligonucleoside Dimer and At Its 3' Terminus By A3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl] LinkedOligonucleoside Dimer

A mixed oligonucleoside-oligonucleotide-oligonucleoside having a3'-de(oxophosphinico)-3'-[methyl(iminooxymethylene)] linkedoligonucleoside dimer and a3'-de(oxyphosphinico)-3'-[(methylimino)-1,2-ethanediyl] linkedoligonucleoside dimer coupled together via a phosphorothioate nucleotidelinkage will be prepared by reacting compound 58, compound 70 andtetrazole in anhydrous acetonitrile under argon. The coupling reactionwill be allowed to proceed to completion followed by treatment withBeaucage reagent and ammonium hydroxide removal of the dimethoxytritylblocking group according to the procedure of Zon, G. and Stec, W. J.,Phosphorothioate oligonucleotides, Oligonucleotides and Analogs APractical Approach, F. Eckstein Ed., IRL Press, pg. 87 (1991). The 3'blocking group will then be removed as per the procedure of Step 3 ofExample 4 and the product purified by HPLC to yield the title compound90, wherein utilizing the structure of Scheme XVI, T₃ and T₅ are OH, Dis S, E is OH, X is H, Q is O, r is 0, and q is 2; and for each q, i.e.,q₁ and q₂, n and p are 1 in each instance;; and for q₁, m is 1; and forq₂, m is 0; and Bxj and Bxi are thymine.

B. Solid Support Synthesis of3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-(3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-(3'→5')-Thymidylyl-)3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-(3'→5')-Thymidylyl-(3'→5')-P-2'-Deoxycytidine,91, A Mixed Oligonucleotide-Oligonucleoside Polymer Incorporating3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)] LinkedOligonucleoside Dimers Flanked By Conventional Linked Nucleotides

The dimeric oligonucleoside 58 will be utilized as building block unitsin a conventional oligonucleotide solid support synthesis as per theprocedure of Example 80. For the purpose of illustration a polymerincorporating seven nucleosides is described. A first unit of thedimeric oligonucleoside 58 will be coupled to a first cytidinenucleoside tethered to a solid support via its 3' hydroxyl group andhaving a free 5' hydroxyl group. After attachment of the first unit ofcompound 58 to the support, the 5'-dimethoxytrityl group of that firstcompound 58 unit will be removed in the normal manner. A second compound58 unit will then be coupled via itsβ-cyanoethyl-N-diisopropylphosphiryl group to the first compound 58 unitusing normal phosphoramidate chemistry. This forms a second compound 58units and elongates the polymer by two nucleosides (or oneoligonucleoside dimer unit). The dimethoxytrityl blocking group from thesecond compound 58 unit will be removed in the normal manner and thepolymer elongated by a further dimeric unit of compound 58. As withaddition of the first and second dimeric units, the third unit ofcompound 58 is coupled to the second via conventional phosphoramiditeprocedures. The addition of the third unit of compound 58 completes thedesired length and base sequence. This polymer has a backbone ofalternating normal phospphodiester linkages and themethyl(iminooxymethylene) linkages of compound 58. The 5'-terminaldimethoxytrityl group of the third compound 58 unit will be removed inthe normal manner followed by release of the polymer from the solidsupport, also in the normal manner. Purification of the polymer will beachieved by HPLC to yield compound 91 wherein, utilizing the structureof Scheme XVI, T₃ and T₅ are OH, D is O, E is OH, X is H, Q is O, r is 1and for the seven nucleoside polymer described, q is 3; and for each q,i.e., q₁, q₂ and q₃, n and p are 1 in each instances; and for q₁ and q₂,m is 1; and for q₃, m is 0; and Bxk is cytosine; and each BxJ and Bxi isthymine.

Example 82 3'-Deoxy-3'-C-formyl-5'-O-t-butyldiphenylsilyl-thymidine

A mixture of thymidine (400 g, and 1.65 mol), 4-dimethylaminopyridine(0.8 g, 6.5 mmol) and t-butyldiphenylchlorosilane (347.2 g, 1.26 mol) inanhydrous pyridine (3.0 lt) was stirred at room temperature for 48 hr.To the stirred reaction mixture two lots of t-butyldiphenyl chlorosilane(129.4 g, 0.47 mol and 22.7 g, 0.082 mol) were added 12 hr. apart andstirring continued for an additional 48 hr. The reaction mixture wasconcentrated under vacuum and the residue redissolved in methanol (2.5lt). The product was precipitated by pouring the reaction mixture into acold stirred ice-water (5.0 lt) suspension. The aqueous suspension wasstirred at room temperature for 3 hr. to quench traces of unreactedchlorosilane. The granular white precipitate was filtered and washedwith distilled water (5×1 lt) and air dried to furnish 876 g of5'-O-t-butyldiphenylsilyl thymidine (slightly contaminated withbissilyated product, about 5%). The impurity was removed by suspendingfinely powdered 5'-O-t-butyldiphenylsilyl thymidine in ether (600 ml)and pouring into stirred hexanes (1.5 lt).

The hexanes:ether slurry was stirred for 1 hr and filtered to furnish5'-O-t-butyldiphenylsilyl thymidine as fine white solid. The product wasfree of bissilyated impurity (judged by t1C; EtOAC:hexanes, 1:1, v/v)and on drying under vacuum furnished 718.9 g (90.7%) of5'-O-t-butyldiphenylsilyl thymidine, which was pure according tothin-layer chromatography. ¹ HNMR (DCl₃) d 1.0 (s, 9 H, t BuH), 1.62 (s,3 H, C5, CH₃), 2.3 (m, 2 H, C2, CH₂), 2.7 (br S, 1 H, 3'OH), 3.8-4.1 (m,3 H, C4, H and (5'CH₈), 4.6 (m, 1 H, 3'H), 6.45 (5, 1 H, 1'H), 7.36-7.67(m, 11 H, (6 H and Ar H), and 9.05 (br S, 1 H, NH).

To a suspension of 5'-O-i-butyldiphenylsilylthymidine (96.0 g, 0.2 mol)in dry toluene (1.1 lt) was added pyridine (19.15 g, 0.24 mol) andN-hydroxysuccimidine (4.6 g, 0.039 mol). The mixture was stirred at 55°C. under arogon while a solution of phenylchlorothioanoformate (38.28 g,0.22 mol in dry toluene, 100 ml) was added dropwise over a period of 1hr. The internal temperature of the reaction mixture rose to 70° C.while it became clear. After 24 hr, the reaction mixture pyridine (1.6g, 0.02 mol) followed by phenylchlorothionoformate (3.48 g, 0.02 mol)were added. The stirring was continued at room temperature for 24 hr.The resulting pyridinium hydrochloride salt was precipitated by additionof ether (400 ml) and filtered again. The filtrate was concentratedunder vacuum and the residue was used for subsequent radical reactionwithout any further purification.

A mixture of the 5'-O-t-butyldiphenylsilyl-3'-O-phenoxythiocarbonyl-thymidine (152.8 g, 0.24 mol),tri-n-butyltin styrene (245 g, 0.62 mol) and aza-bis(isobutyronitrile)(5.95 g, 0.036 mol) in dry benzene (800 ml) were degassed with argon (3times) and heated at 75° C. for 8 hr while stirring. Over next 60 hr,AlBN (6×5.95 g), 0.036 mol) was added in portions to the reactionmixture under argon and stirring was continued at 75° C. Aftercompletion of the reaction (about 70-80 hr; detected by completeconsumption of the 5'-O-t-butyldiphenylsilyl-3'-O-phenoxythiocarbonyl-thymidine), the solution wascooled to room temperature and transferred on the top of a prepackedsilica gel (1 mg) column. Elution with EtOAC;Hexanes (7:5, v/v) gave thedesired 3'-styryl nucleoside as homogenous material. Appropriatefractions were pooled and evaporated to furnish 67.49 (49.5%) of the3'-styryl nucleoside as an oil. ¹ HNMR (CDCl₃) d 1.1 (S, 9 H, tBu-H),1.60 (S, 3 H, C₅ CH₃), 2.4 (m, 2 H, C₂ CH₂) 3.25 (m, 1 H, C₃ H), 3.8 (m,1 H, C4'H), 4.15 (m, 2 H, (₅, CH₂), 6.21 (dd, 1, C₁, H), 6.0 and 6.5 (2m, 2 H, CH═CH-ph), 7.3-7.7 (m, 11 H, C₆ H, and Ar H), 8.8 (S, 1 H, OH).

A mixture of the 3'-styryl nucleoside (2.19 g, 3.86 mmol), N-methylmorpholine-N-oxide (0.68 g, 5.6 mmol), OsO₄ (3.9 ml of 2.5% solution int-BuOH, 0.38 mmol) in dioxane: water (30 ml, 2:1) was stirred at roomtemperature. The reaction mixture was protected from light and stirredfor 1 hr. To the dark colored reaction mixture NaIO₄ (1.82 g, 8.5 mmol)in water (8 ml) was added in one portion and stirring continued for 3hr. After completion of the reaction, the reaction mixture was dilutedwith EtOAC (100 ml) and extracted with saturated NaCl solution (3×60ml). The organic layer was dried (MgSO₄) and concentrated to furnishoily residue. The residue was purified by silica gel columnchromatograpy to furnish 0.94 g (50%) of5'-O-t-butyldiphenylsilyl-3'-c-formylthymidine as white foam. ¹ HNMR(CDCl₃) d 1.1 (s, 9 H, t-BuH), 1.61 (S, 3 H, C₅ CH₃), 2.3 and 2.75 (2 m,2 H, C2, CH₂), 3.4 (m, 1 H, C_(3') H), 4.0 (m, 2 H, C_(5') CH₂), 4.35(m, 1 H, C_(4') H), 6.11 (t, 1, C_(1') H), 7.26-7.67 (m, 11 H, C₆ H, ArH), 8.2 (brS, 1 H, NH), and 9.70 (s, 1 H, CHO).

EVALUATION Procedure 1--Nuclease Resistance

A. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to serum and cytoplasmic nucleases.

Oligonucleotide-mimicking macromolecules of the invention can beassessed for their resistance to serum nucleases by incubation of theoligonucleotide-mimicking macromolecules in media containing variousconcentrations of fetal calf serum or adult human serum. Labelledoligonucleotide-mimicking macromolecules are incubated for varioustimes, treated with protease K and then analyzed by gel electrophoresison 20% polyacrylamine-urea denaturing gels and subsequentautoradiography. Autoradiograms are quantitated by laser densitometry.Based upon the location of the modified linkage and the known length ofthe oligonucleotide-mimicking macromolecules it is possible to determinethe effect on nuclease degradation by the particular modification. Forthe cytoplasmic nucleases, an HL 60 cell line can be used. Apost-mitochondrial supernatant is prepared by differentialcentrifugation and the labelled macromolecules are incubated in thissupernatant for various times. Following the incubation, macromoleculesare assessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated for evaluation ofthe macromolecules of the invention. It is expected that themacromolecules will be completely resistant to serum and cytoplasmicnucleases.

B. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to specific endo- and exo-nucleases.

Evaluation of the resistance of natural oligonucleotides andoligonucleotide-mimicking macromolecules of the invention to specificnucleases (i.e., endonucleases, 3',5'-exo-, and 5',3'-exonucleases) canbe done to determine the exact effect of the macromolecule linkage ondegradation. The oligonucleotide-mimicking macromolecules are incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea is added andanalysis on 20% polyacrylamide gels containing urea is done. Gelproducts are visualized by staining with Stains All reagent (SigmaChemical Co.). Laser densitometry is used to quantitate the extent ofdegradation. The effects of the macromolecules linkage are determinedfor specific nucleases and compared with the results obtained from theserum and cytoplasmic systems. As with the serum and cytoplasmicnucleases, it is expected that the oligonucleotide-mimickingmacromolecules of the invention will be completely resistant to endo-and exo-nucleases.

Procedure 2--5-Lipoxygenase Analysis and Assays

B. Research Reagents

The oligonucleotide-mimicking macromolecules of this invention will alsobe useful as research reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 30° toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

B. Diagnostics

The oligonucleotide-mimicking macromolecules of the invention will alsobe useful in diagnostic applications, particularly for the determinationof the expression of specific mRNA species in various tissues or theexpression of abnormal or mutant RNA species. In this example, while themacromolecules target a abnormal mRNA by being designed complementary tothe abnormal sequence, they would not hybridize to normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standardmethods. The crude homogenate or extract can be treated for example toeffect cleavage of the target RNA. The produce can then be hybridized toa solid support which contains a bound oligonucleotide complementary toa region on the 5' region of the mRNA would bind to the solid support.The 3' region of the abnormal RNA, which is cleaved, would not be boundto the support and therefore would be separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

C. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotide-mimicking macromolecules whichinterfere with 5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotide-mimicking macromolecules makesuse of the fact that 5-lipoxygenase is a "suicide" enzyme in that itinactivates itself upon reacting with substrate. Treatment ofdifferentiated HL-60 or other cells expressing 5 lipoxygenase, with 10μM A23187, a calcium ionophore, promotes translocation of 5-lipoxygenasefrom the cytosol to the membrane with subsequent activation of theenzyme. Following activation and several rounds of catalysis, the enzymebecomes catalytically inactive. Thus, treatment of the cells withcalcium ionophore inactivates endogenous 5-lipoxygenase. It takes thecells approximately 24 hours to recover from A23187 treatment asmeasured by their ability to synthesize leukotriene B₄. Macromoleculesdirected against 5-lipoxygenase can be tested for activity in two HL-60model systems using the following quantitative assays. The assays aredescribed from the most direct measurement of inhibition of5-lipoxygenase protein synthesis in intact cells to more downstreamevents such as measurement of 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotide-mimicking macromolecules can exerton intact cells and which can be easily be quantitated is specificinhibition of 5-lipoxygenase protein synthesis. To perform thistechnique, cells can be labelled with ³⁵ S-methionine (50 μCi/mL) for 2hours at 37° C. to label newly synthesized protein. Cells are extractedto solubilize total cellular proteins and 5-lipoxygenase isimmunoprecipitated with 5-lipoxygenase antibody followed by elution fromprotein A Sepharose beads. The immunoprecipitated proteins are resolvedby SDS-polyacrylamide gel electrophoresis and exposed forautoradiography. The amount of immunoprecipitated 5-lipoxygenase isquantitated by scanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM ¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mlphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 pmolarachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide-mimicking macromolecule would oxidize195 pmol, 140 pmol, and 60 pmol of arachidonic acid/5 min/10⁶ cellsrespectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC in usedas a standard and as the primary antigen to coat microtiter plates. 25ng of purified 5-lipoxygenase is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris•HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labelled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 meroligonucleotide-mimicking macromolecule at 1 μM, 10 μM, and 30 μM wouldbe 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10⁶ cells, respectivelywith untreated cells containing about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophiol-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide-mimicking macromolecule directed to the 5-LO mRNAl.Cells will be treated for 72 hours with either 1 μM, 10 μM or 30 μM ofthe macromolecule in the presence of 1.3% DMSO. The quantity of LTB₄produced from 5×10⁵ cells would be expected to be about 75 pg, 50 pg,and 35 pg, respectively with untreated differentiated cells producing 75pg LTB₄.

D. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotide-mimicking macromolecules will be appliedtopically to both ears 12 to 24 hours prior to administration ofarachidonic acid to allow optimal activity of the compounds. Both earsare pretreated for 24 hours with either 0.1 μmol, 0.3 μmol, or 1.0 μmolof the macromolecule prior to challenge with arachidonic acid. Valuesare expressed as the means for three animals per concentration.Inhibition of polymorphonuclear cell infiltration for 0.1 μmol, 0.3μmol, and 1 μmol is expected to be about 10%, 75% and 92% of controlactivity, respectively. Inhibition of edema is expected to be about 3%,58% and 90%, respectively while inhibition of leukotriene B₄ productionwould be expected to be about 15%, 79% and 99%, respectively.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A method for forming between adjacent sugarmoieties a covalent linkage having structure CH═N--R_(A) --CH₂, CH₂--R_(A) --N═CH, or R_(A) --N═CH--CH₂ where R_(A) is O or NR₁, comprisingthe steps of:(a) providing synthons having structure: ##STR4## (b)contacting said synthons for a time and under reaction conditionseffective to form said covalent linkage;wherein: Z₁ and Y₂ are selectedsuch that(i) Z₁ is C(O)H and Y₂ is CH₂ R_(A) NH₂ ; or (ii) Z₁ is CH₂R_(A) NH₂ and Y₂ is C(O)H; or (iii) Z₁ is R_(A) NH₂ and Y₂ is H(O)CCH₂ ;R₁ is H or alkyl having 1 to about 10 carbon atoms; B_(X1) and B_(X2)are, independently, nucleosidic bases; Q₁ and Q₂ are O; and X₁ and X₂are, independently, H; OH; F; or O-alkyl having 1 to about 10 carbonatoms.
 2. The method of claim 1 wherein R_(A) is O.
 3. The method ofclaim 1 wherein R_(A) is NH or NCH₃.
 4. The method of claim 1 wherein Z₁is C(O)H and Y₂ is CH₂ R_(A) NH₂.
 5. The method of claim 1 wherein Z₁ isCH₂ R_(A) NH₂ and Y₂ is C(O)H.
 6. The method of claim 1 wherein Z₁ isR_(A) NH₂ and Y₂ is H(O)CCH₂.
 7. The method of claim 1 wherein at leastone of X₁ and X₂ is H.
 8. The method of claim 1 wherein at least one ofX₁ and X₂ is OH.
 9. The method of claim 1 wherein at least one of X₁ andX₂ is F.
 10. The method of claim 1 wherein at least one of X₁ and X₂ isO-alkyl.
 11. A method for forming between adjacent sugar moieties acovalent linkage having structure CH₂ --NR₁ --R_(A) --CH₂, CH₂ --R_(A)--NR₁ --CH₂, R_(A) --NR₁ --CH₂ --CH₂ where R_(A) is O or NR₂, comprisingthe steps of:(a) providing synthons having structures: ##STR5## (b)contacting said synthons for a time and under reaction conditionseffective to form an intermediate linkage; and (c) reducing saidintermediate linkage to form said covalent linkage;wherein: Z₁ and Y₂are selected such that(i) Z₁ is C(O)H and Y₂ is CH₂ R_(A) NH₂ ; or (ii)Z₁ is CH₂ R_(A) NH₂ and Y₂ is C(O)H; or (iii) Z₁ is R_(A) NH₂ and Y₂ isH(O)CCH₂ ; R₁ and R₂ are, independently, H or alkyl having 1 to about 10carbon atoms; B_(X1) and B_(X2) are, independently, nucleosidic bases;Q₁ and Q₂ are O; and X₁ and X₂ are, independently, H; OH; F; or O-alkylhaving 1 to about 10 carbon atoms.
 12. The method of claim 11 whereinR_(A) is O.
 13. The method of claim 11 wherein R_(A) is NH or NCH₃. 14.The method of claim 11 wherein Z₁ is C(O)H and Y₂ is CH₂ R_(A) NH₂. 15.The method of claim 11 wherein Z₁ is CH₂ R_(A) NH₂ and Y₂ is C(O)H. 16.The method of claim 11 wherein Z₁ is R_(A) NH₂ and Y₂ is H(O)CCH₂. 17.The method of claim 11 wherein at least one of X₁ and X₂ is H.
 18. Themethod of claim 11 wherein at least one of X₁ and X₂ is OH.
 19. Themethod of claim 11 wherein at least one of X₁ and X₂ is F.
 20. Themethod of claim 11 wherein at least one of X₁ and X₂ is O-alkyl.